Focusing methods and optical systems and assemblies using the same

ABSTRACT

A method for controlling a focus of an optical system. The method includes providing a pair of incident light beams to a conjugate lens. The incident light beams are directed by the lens to converge toward a focal region. The method also includes reflecting the incident light beams with an object positioned proximate to the focal region. The reflected light beams return to and propagate through the lens. The method also includes determining relative separation measured between the reflected light beams and determining a degree-of-focus of the optical system with respect to the sample based upon the relative separation.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/300,300, filed Feb. 1, 2010 and entitled “FocusingMethods and Optical Systems and Assemblies Using the Same,” which isincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Embodiments of the present invention relate generally to optical systemsand assemblies, and more specifically to focusing methods formicroscopic optical systems and assemblies.

A wide variety of microscopic optical systems exist that observe asample of interest comprising biological or chemical substances. Forexample, sample imagers may be configured to detect activity that isindicative of a desired reaction (e.g., binding events between targetsand probes). Such activity may be identified by detecting lightemissions (e.g., fluorescence or chemiluminescence) from labels that areselectively bound to the targets or probes. The detected light is thenanalyzed to determine properties or characteristics of the biological orchemical substances. Other microscopic optical systems exist that areconfigured to inspect an object to determine certain features orstructures of the object. For example, optical systems may be used toinspect a surface of a semiconductor chip or silicon wafer to determinewhether there are any deviations or defects in a pattern on the surface.Other optical systems include profilometers that determine surfaceprofiles of an object.

Conventional optical systems, such as those described above, generallyinclude a focus-control system that determines whether the opticalsystem has an acceptable degree-of-focus with respect to the object. Forexample, some conventional optical systems use a focusing method thatincludes reflecting a reference light beam off a surface of the objectand detecting the reflected light beam with a detector (e.g.,position-sensitive detector (PSD)). The reflected light beam forms abeam spot on a surface of the detector. If the beam spot is offset by acertain amount from a desired location on the surface or if the beamspot has a certain morphology (e.g., size, shape, and/or density), thefocus-control system may determine that the optical system is notproperly focused and may adjust the object or the optical components ofthe system accordingly.

However, the focus-control systems of such conventional optical systemshave certain limitations. Focus-control systems often include severaloptical components that affect the optical path of the reference lightbeam before and after the light beam is reflected by the object. If anyone of these optical components is somehow moved from a predeterminedposition during operation of the optical system or somehow adverselyaffected, the beam spot will not provide accurate information relatingto the focus of the system. Such problems may not be identified untilafter an object is scanned thereby requiring the use of sub-standarddata or possibly requiring another scan. In some cases, acquisition ofanother scan may not be possible and a valuable sample can end up beingwasted. It may also be necessary to recalibrate the optical componentsof the focus-control system, which may take substantial time and coststo remedy. Sub-standard data, loss of samples, or time wasted inobtaining data can be particularly problematic in diagnostic orprognostic applications where samples are often scarce and the dataprovides information that is important in determining a course oftreatment for a patient.

In addition to the above, conventional optical systems may use complexbeam-spot analysis algorithms to analyze the location, shape, anddensity of the beam spot. Such analysis may be costly and also sensitiveto the configuration of the optical components.

Accordingly, there is a need for focusing methods and focus-controlsystems that reduce the alignment sensitivity of the optical components.Furthermore, there is a need for focus-control systems that usealternative forms of beam-spot analysis. There is also a general needfor improved focusing methods and focus-control systems that aresimpler, more accurate, and/or less costly than known focusing methodsand focus-control systems.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with one embodiment, a method for controlling a focus ofan optical system is provided. The method includes providing a pair ofincident light beams to a conjugate lens. The incident light beams aredirected by the lens to converge toward a focal region. The method alsoincludes reflecting the incident light beams with an object positionedproximate to the focal region. The reflected light beams return to andpropagate through the lens. The method further includes determining arelative separation between the reflected light beams and determining adegree-of-focus of the optical system with respect to the object basedupon the relative separation. The method can be particularly useful foran optical system that is configured for biological or chemicalanalysis, for example, at microscopic resolution.

In another embodiment, an optical system is provided. The imagerincludes a light source that is configured to provide a pair of incidentlight beams and a conjugate lens positioned to receive the incidentlight beams. The lens directs the incident light beams toward a focalregion. The imager also includes an object holder that is configured tohold an object with respect to the focal region. The object reflects theincident light beams so that the reflected light beams return to andpropagate through the lens. The imager also includes a focus-controlsystem that is configured to determine a relative separation between thereflected light beams and determine a degree-of-focus based upon therelative separation. The imager can be configured for biological orchemical analysis, for example, at microscopic resolution.

In yet another embodiment, a method for controlling a focus of anoptical system is provided. The method includes providing a pair ofincident light beams to a conjugate lens. The incident light beams aredirected by the lens to converge toward a focal region. The method alsoincludes reflecting the incident light beams with an object that ispositioned proximate to the focal region. The reflected light beamsreturn to and propagating through the lens. The method also includesdetermining a phase of each of the reflected light beams and determininga degree-of-focus of the optical system with respect to the sample bycomparing the phases of the reflected light beams. The method can beparticularly useful for a sample imager that is configured forbiological or chemical analysis, for example, at microscopic resolution.

In a further embodiment, an optical system is provided. The imagerincludes a light source that is configured to provide a pair of incidentlight beams and a conjugate lens that is positioned to receive theincident light beams. The lens directs the incident light beams toward afocal region. The optical system also includes an object holder that isconfigured to hold an object with respect to the focal region. Thesample reflects the incident light beams so that the reflected lightbeams return to and propagate through the lens. The imager also includesa focus-control system that is configured to determine a phase of eachof the reflected light beams and determine a degree-of-focus bycomparing the phases of the reflected light beams. The imager can beconfigured for biological or chemical analysis, for example, atmicroscopic resolution.

Also provided is a method for determining distance of an object from alens. The method includes providing a pair of incident light beams to aconjugate lens. The incident light beams are directed by the lens toconverge toward a focal region. The method also includes reflecting theincident light beams with an object positioned at a working distanceproximate to the focal region. The reflected light beams return to andpropagate through the lens. The method further includes determining aseparation distance measured between the reflected light beams anddetermining the working distance between the object and the lens basedupon the separation distance. The method can be particularly useful fora sample imager that is configured for biological or chemical analysis,for example, at microscopic resolution.

In another embodiment, a sample imager is provided. The imager includesa light source that is configured to provide a pair of incident lightbeams and a conjugate lens positioned to receive the incident lightbeams 1. The lens directs the incident light beams to a focal region.The imager also includes a sample holder that is configured to hold asample at a working distance from the lens. The sample reflects theincident light beams so that the reflected light beams return to andpropagate through the lens. The imager also includes a system that isconfigured to determine a separation distance measured between thereflected light beams and determine the working distance based upon theseparation distance. The sample imager can be configured for a varietyof samples or objects including, for example, those used in biologicalor chemical analyses. The imager can be configured for microscopicresolution.

In yet another embodiment, a method for determining distance of anobject from a lens is provided. The method includes providing a pair ofincident light beams to a conjugate lens. The incident light beams aredirected by the lens to converge toward a focal region. The method alsoincludes reflecting the incident light beams with an object that ispositioned at a working distance proximate to the focal region. Thereflected light beams return to and propagating through the lens. Themethod also includes determining a phase of each of the reflected lightbeams and determining the working distance by comparing the phases ofthe reflected light beams. The method can be particularly useful for asample imager that is configured for biological or chemical analysis,for example, at microscopic resolution.

In a further embodiment, a sample imager is provided. The imagerincludes a light source that is configured to provide a pair of incidentlight beams and a conjugate lens that is positioned to receive theincident light beams. The lens directs the incident light beams to afocal region. The sample imager also includes a sample holder that isconfigured to hold a sample with respect to the focal region. The samplereflects the incident light beams so that the reflected light beamsreturn to and propagate through the lens. The imager also includes asystem that is configured to determine a phase of each of the reflectedlight beams and determine the working distance by comparing the phasesof the reflected light beams. The sample imager can be configured for avariety of samples or objects including, for example, those used inbiological or chemical analyses. The imager can be configured formicroscopic resolution.

In another embodiment, a method of determining a profile of an objectsurface is provided. The method includes providing a pair of incidentlight beams to a conjugate lens. The incident light beams are directedby the lens to converge toward a focal region. The method also includesreflecting the incident light beams with an object surface of an objectthat is positioned at a working distance from the lens proximate to thefocal region. The reflected light beams returning to and propagatingthrough the lens. The method also includes monitoring relativeseparation between the reflected light beams while scanning the objectsurface. The relative separation changes when the working distancechanges. The method also includes determining a profile of the objectsurface based upon the relative separation monitored along the objectsurface. The method can be particularly useful for an optical system,such as a profilometer, that determines a surface profile of an object.

In another embodiment, a method for controlling a focus of an opticalsystem is provided. The method includes providing first and secondparallel incident light beams to a conjugate lens. The incident lightbeams are directed by the lens to converge toward a focal region. Themethod also includes reflecting the incident light beams with an objectpositioned proximate to the focal region. The reflected light beamsreturn to and propagate through the lens. The incident light beams havea projection relationship. The method also includes reflecting the firstand second light beams with a plurality of optical components configuredto maintain the projection relationship of the incident light beams.

In another embodiment, a method of determining an angle of an objectsurface is provided. The method includes providing at least two pairs ofincident light beams to a conjugate lens. A first pair of incident lightbeams are directed by the lens to converge toward a first focal regionand a second pair of incident light beams are directed by the lens toconverge toward a second focal region. The method also includesreflecting the at least two pairs of incident light beams with an objectsurface of an object that is positioned at a working distance from thelens proximate to the first focal region and the second focal region. Afirst pair of reflected light beams and a second pair of reflected lightbeams return to and propagate through the lens. The method also includesdetermining relative separation between the first pair of reflectedlight beams and determining relative separation between the second pairof reflected light beams. The relative separation changing when theworking distance changes. The method further includes determining theangle of the object surface based upon the relative separation betweenthe first pair of reflected light beams and based upon the relativeseparation between the second pair of reflected light beams.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a block diagram of an optical system formed inaccordance with an embodiment.

FIG. 2 is a perspective view of an optical assembly formed in accordancewith one embodiment that may be used with the optical system shown inFIG. 1.

FIG. 3 is a plan view of the optical assembly shown in FIG. 2.

FIG. 4 illustrates incident and reflected light beams when the opticalassembly shown in FIG. 2 is in focus with respect to an object.

FIG. 5 illustrates beam spots on a detector surface that are provided bythe reflected light beams shown in FIG. 4.

FIG. 6 illustrates incident and reflected light beams when the opticalassembly shown in FIG. 2 is below focus.

FIG. 7 illustrates beam spots on a detector surface that are provided bythe reflected light beams shown in FIG. 6.

FIG. 8 illustrates incident and reflected light beams when the opticalassembly shown in FIG. 2 is above focus.

FIG. 9 illustrates beam spots on a detector surface that are provided bythe reflected light beams shown in FIG. 8.

FIG. 10 illustrates alternative embodiments for determining relativeseparation between reflected light beams.

FIG. 11 illustrates reflection of parallel reflected light beams by twooptical components where one of the optical components is not in adesired position.

FIG. 12 illustrates beam spots on a detector surface that are providedby the reflected light beams shown in FIG. 11.

FIG. 13 illustrates reflection of parallel reflected light beams by twooptical components where one of the optical components is not in adesired position.

FIG. 14 illustrates beam spots on a detector surface that are providedby the reflected light beams shown in FIG. 13.

FIG. 15 is a side view of a flow cell that may be used in variousembodiments and illustrates reflection of incident light beams.

FIGS. 16A and 16B are schematic diagrams of optical assemblies that maybe formed in accordance with alternative embodiments.

FIG. 17 is a perspective view of an optical assembly formed inaccordance with another embodiment that may be used with the opticalsystem shown in FIG. 1.

FIG. 18 is a plan view of the optical assembly shown in FIG. 17.

FIG. 19 is a side view of a beam folding device that may be used inaccordance with various embodiments.

FIGS. 20-22 illustrate an object being scanned by an optical assemblyformed in accordance with various embodiments.

FIG. 23 is a perspective view of a sample imager formed in accordancewith one embodiment.

FIG. 24 is a block diagram that illustrates a method of determining adegree-of-focus of an object with respect to an optical assembly.

FIG. 25 is a block diagram illustrating a control loop for controlling adegree-of-focus of an optical system with respect to an object orsample.

FIG. 26 is a block diagram that illustrates a method of determining aworking distance between an object and a conjugate lens of an opticalassembly.

FIG. 27 is a block diagram illustrating a control loop for profiling anobject surface.

FIG. 28 is a block diagram that illustrates a method of operating anoptical system in accordance with various embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments described herein include optical systems that may be used toat least one of view, image, and inspect various objects. In someembodiments, the optical systems include sample imagers that are used toimage samples for biological or chemical analysis. For example, a sampleimager may be configured to perform at least one oftotal-internal-reflectance-fluorescence (TIRF) imaging andepi-fluorescent imaging. In particular embodiments, the sample imager isa scanning time-delay integration (TDI) system. In other embodiments,the optical systems may be configured to inspect surfaces ofmicrodevices, such as semi-conductor chips or silicon wafers, todetermine if the surfaces have any deviations or defects. In otherembodiments, the optical systems include profilometers that areconfigured to determine a surface profile or topography of an object.

In various embodiments, the optical systems can include a conjugate lensthat receives one or more pairs of parallel incident light beams. Theconjugate lens can direct the incident light beams to a focal regionwhere the incident light beams are reflected by a surface or interfaceof an object that is proximate to the focal region. The conjugate lensmay then receive the reflected light beams. If the object is in-focus,the reflected light beams will project parallel to one another from theconjugate lens. If the object is not in-focus, the reflected light beamswill project from the conjugate lens in a non-parallel manner.

Optical systems described herein may determine a focus or profileparameter that is effectively based upon or at least partiallydetermined by the projection relationship of the reflected light beamsexiting the conjugate lens. The focus or profile parameter may be afunction of one or more geometric characteristics or elements of areflected light beam, such as an optical path length or projection anglefrom an optical component. Furthermore, the parameter may be a functionof a relation between the reflected light beams (ratio of geometriccharacteristics, separation distance between beam spots, path spacingbetween beams). Focus and profile parameters may be indicative of adegree-of-focus of the optical system, a working distance that separatesthe lens and the object, or a surface profile (e.g., height) of theobject at a particular point on the object surface.

By way of example, as the two non-parallel reflected light beamspropagate along an optical track, a distance that separates thereflected light beams increases or decreases. Optical systems describedherein may determine a relative separation between the reflected lightbeams. The relative separation is based upon or at least partiallydetermined by the projection relationship of the reflected light beams.For example, the light beams may be propagating parallel to each otherthereby maintaining the relative separation, converging toward eachother thereby decreasing the relative separation, or diverging away fromeach other thereby increasing the relative separation. The relativeseparation may be determined, for example, as a separation distance thatextends between beam spots on a detector's surface. The relativeseparation may also be calculated by individually detecting the beamspots with different detectors. The relative separation can be used todetermine a working distance that extends between the conjugate lens andthe object that reflected the incident light beams. The relativeseparation may also be used to determine a degree-of-focus of theoptical system. Furthermore, the relative separation may be used todetermine a surface profile of the object.

Furthermore, as will be described in greater detail below, embodimentsdescribed herein include one or more focus-control systems to determinewhether the object, which may include a sample, is sufficiently within afocal plane of the optical system so that the object can be viewed,imaged, and/or inspected. More specifically, embodiments can determinewhether the optical system has a sufficient degree-of-focus with respectto the object. The focus determination may be based on reference lightbeams that are incident upon the object. After determining whether theobject is sufficiently in focus with respect to the optical system,embodiments can automatically move the object or the optical system sothat the object is within the focal plane of the optical system.

As used herein, the term “object” includes all things that are suitablefor imaging, viewing, analyzing, inspecting, or profiling with theoptical systems described herein. By way of example only, objects mayinclude semiconductor wafers or chips, recordable media, samples, flowcells, microparticles, slides, or microarrays. Objects generally includeone or more surfaces and/or one or more interfaces that a user maydesire to image, view, analyze, inspect, and/or determine a profilethereof. The objects may have surfaces or interfaces with relieffeatures such as wells, pits, ridges, bumps, beads or the like.

As used herein, the term “sample” includes various matters of interest.A sample may be imaged or scanned for subsequent analysis. In particularembodiments, a sample may include biological or chemical substances ofinterests and, optionally, an optical substrate that supports thebiological or chemical substances. As such, a sample may or may notinclude an optical substrate. As used herein, the term “biological orchemical substances” is not intended to be limiting, but may include avariety of biological or chemical substances that are suitable for beingimaged or examined with the optical systems described herein. Forexample, biological or chemical substances include biomolecules, such asnucleosides, nucleic acids, polynucleotides, oligonucleotides, proteins,enzymes, polypeptides, antibodies, antigens, ligands, receptors,polysaccharide, carbohydrate, polyphosphates, nanopores, organelles,lipid layers, cells, tissues, organisms, and biologically activechemical compound(s) such as analogs or mimetics of the aforementionedspecies.

The biological or chemical substances may be supported by an opticalsubstrate. As used herein, the term “optical substrate” is not intendedto be limiting, but may include various materials that support thebiological or chemical substances and permit the biological or chemicalsubstances to be at least one of viewed, imaged, and examined. Forexample, the optical substrate may comprise a transparent material thatreflects a portion of incident light and refracts a portion of theincident light. Alternatively, the optical substrate may be, forexample, a mirror that reflects the incident light entirely such that nolight is transmitted through the optical substrate. Typically, theoptical substrate has a flat surface. However, the optical substrate canhave a surface with relief features such as wells, pits, ridges, bumps,beads or the like.

In an exemplary embodiment, the optical substrate is a flow cell havingflow channels where nucleic acids are sequenced. However, in alternativeembodiments, the optical substrate may include one or more slides,planar chips (such as those used in microarrays), or microparticles. Insuch cases where the optical substrate includes a plurality ofmicroparticles that support the biological or chemical substances, themicroparticles may be held by another optical substrate, such as a slideor grooved plate. In particular embodiments, the optical substrateincludes diffraction grating based encoded optical identificationelements similar to or the same as those described in pending U.S.patent application Ser. No. 10/661,234, entitled Diffraction GratingBased Optical Identification Element, filed Sep. 12, 2003, which isincorporated herein by reference in its entirety, discussed morehereinafter. A bead cell or plate for holding the optical identificationelements may be similar to or the same as that described in pending U.S.patent application Ser. No. 10/661,836, entitled “Method and Apparatusfor Aligning Microbeads in Order to Interrogate the Same”, filed Sep.12, 2003, and U.S. Pat. No. 7,164,533, entitled “Hybrid Random Bead/ChipBased Microarray”, issued Jan. 16, 2007, as well as U.S. patentapplication Ser. No. 60/609,583, entitled “Improved Method and Apparatusfor Aligning Microbeads in Order to Interrogate the Same”, filed Sep.13, 2004, Ser. No. 60/610,910, entitled “Method and Apparatus forAligning Microbeads in Order to Interrogate the Same”, filed Sep. 17,2004, each of which is incorporated herein by reference in its entirety.

As used herein, the term “optical components” or “focus components”includes various elements that affect the transmission of light. Opticalcomponents may be, for example, reflectors, dichroics, beam splitters,collimators, lenses, filters, wedges, prisms, mirrors, and the like.

By way of example, optical systems described herein may be constructedto include various components and assemblies as described in PCTapplication PCT/US07/07991, entitled “System and Devices for Sequence bySynthesis Analysis”, filed Mar. 30, 2007 and/or to include variouscomponents and assemblies as described in PCT applicationPCT/US2008/077850, entitled “Fluorescence Excitation and DetectionSystem and Method”, filed Sep. 26, 2008, both of which the completesubject matter are incorporated herein by reference in their entirety.In particular embodiments, optical systems can include variouscomponents and assemblies as described in U.S. Pat. No. 7,329,860, ofwhich the complete subject matter is incorporated herein by reference inits entirety. Optical systems can also include various components andassemblies as described in U.S. patent application Ser. No. 12/638,770,filed on Dec. 15, 2009, of which the complete subject matter isincorporated herein by reference in its entirety.

In particular embodiments, methods, and optical systems described hereinmay be used for sequencing nucleic acids. For example,sequencing-by-synthesis (SBS) protocols are particularly applicable. InSBS, a plurality of fluorescently labeled modified nucleotides are usedto sequence dense clusters of amplified DNA (possibly millions ofclusters) present on the surface of an optical substrate (e.g., asurface that at least partially defines a channel in a flow cell). Theflow cells may contain nucleic acid samples for sequencing where theflow cells are placed within the appropriate flow cell holders. Thesamples for sequencing can take the form of single nucleic acidmolecules that are separated from each other so as to be individuallyresolvable, amplified populations of a nucleic acid molecules in theform of clusters or other features, or beads that are attached to one ormore molecules of nucleic acid. The nucleic acids can be prepared suchthat they comprise an oligonucleotide primer adjacent to an unknowntarget sequence. To initiate the first SBS sequencing cycle, one or moredifferently labeled nucleotides, and DNA polymerase, etc., can be flowedinto/through the flow cell by a fluid flow subsystem (not shown). Eithera single type of nucleotide can be added at a time, or the nucleotidesused in the sequencing procedure can be specially designed to possess areversible termination property, thus allowing each cycle of thesequencing reaction to occur simultaneously in the presence of severaltypes of labeled nucleotides (e.g. A, C, T, G). The nucleotides caninclude detectable label moieties such as fluorophores. Where the fournucleotides are mixed together, the polymerase is able to select thecorrect base to incorporate and each sequence is extended by a singlebase. One or more lasers may excite the nucleic acids and inducefluorescence. The fluorescence emitted from the nucleic acids is basedupon the fluorophores of the incorporated base, and differentfluorophores may emit different wavelengths of emission light. Exemplarysequencing methods are described, for example, in Bentley et al., Nature456:53-59 (2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO 91/06678; WO07/123,744; U.S. Pat. No. 7,329,492; U.S. Pat. No. 7,211,414; U.S. Pat.No. 7,315,019; U.S. Pat. No. 7,405,281, and US 2008/0108082, each ofwhich is incorporated herein by reference.

Other sequencing techniques that are applicable for use of the methodsand systems set forth herein are pyrosequencing, nanopore sequencing,and sequencing by ligation. Exemplary pyrosequencing techniques andsamples that are particularly useful are described in U.S. Pat. No.6,210,891; U.S. Pat. No. 6,258,568; U.S. Pat. No. 6,274,320 and Ronaghi,Genome Research 11:3-11 (2001), each of which is incorporated herein byreference. Exemplary nanopore techniques and samples that are alsouseful are described in Deamer et al., Acc. Chem. Res. 35:817-825(2002); Li et al., Nat. Mater. 2:611-615 (2003); Soni et al., Clin Chem.53:1996-2001 (2007) Healy et al., Nanomed. 2:459-481 (2007) and Cockroftet al., J. am. Chem. Soc. 130:818-820; and U.S. Pat. No. 7,001,792, eachof which is incorporated herein by reference. Any of a variety ofsamples can be used in these systems such as substrates having beadsgenerated by emulsion PCR, substrates having zero-mode waveguides,substrates having biological nanopores in lipid bilayers, solid-statesubstrates having synthetic nanopores, and others known in the art. Suchsamples are described in the context of various sequencing techniques inthe references cited above and further in US 2005/0042648; US2005/0079510; US 2005/0130173; and WO 05/010145, each of which isincorporated herein by reference.

In other embodiments, optical systems described herein may be utilizedfor detection of samples that include microarrays. A microarray mayinclude a population of different probe molecules that are attached toone or more substrates such that the different probe molecules can bedifferentiated from each other according to relative location. An arraycan include different probe molecules, or populations of the probemolecules, that are each located at a different addressable location ona substrate. Alternatively, a microarray can include separate opticalsubstrates, such as beads, each bearing a different probe molecule, orpopulation of the probe molecules, that can be identified according tothe locations of the optical substrates on a surface to which thesubstrates are attached or according to the locations of the substratesin a liquid. Exemplary arrays in which separate substrates are locatedon a surface include, without limitation, a Sentrix® Array or Sentrix®BeadChip Array available from Inc. (San Diego, Calif.) or othersincluding beads in wells such as those described in U.S. Pat. Nos.6,266,459, 6,355,431, 6,770,441, and 6,859,570; and PCT Publication No.WO 00/63437, each of which is hereby incorporated by reference. Otherarrays having particles on a surface include those set forth in US2005/0227252; WO 05/033681; and WO 04/024328, each of which is herebyincorporated by reference.

Any of a variety of microarrays known in the art, including, forexample, those set forth herein, can be used in embodiments of theinvention. A typical microarray contains sites, sometimes referred to asfeatures, each having a population of probes. The population of probesat each site is typically homogenous having a single species of probe,but in some embodiments the populations can each be heterogeneous. Sitesor features of an array are typically discrete, being separated withspaces between each other. The size of the probe sites and/or spacingbetween the sites can vary such that arrays can be high density, mediumdensity or lower density. High density arrays are characterized ashaving sites separated by less than about 15 μm. Medium density arrayshave sites separated by about 15 to 30 μm, while low density arrays havesites separated by greater than 30 μm. An array useful in the inventioncan have sites that are separated by less than 100 μm, 50 μm, 10 μm, 5μm, 1 μm, or 0.5 μm. An apparatus or method of an embodiment of theinvention can be used to image an array at a resolution sufficient todistinguish sites at the above densities or density ranges.

Further examples of commercially available microarrays that can be usedinclude, for example, an Affymetrix® GeneChip® microarray or othermicroarray synthesized in accordance with techniques sometimes referredto as VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis)technologies as described, for example, in U.S. Pat. Nos. 5,324,633;5,744,305; 5,451,683; 5,482,867; 5,491,074; 5,624,711; 5,795,716;5,831,070; 5,856,101; 5,858,659; 5,874,219; 5,968,740; 5,974,164;5,981,185; 5,981,956; 6,025,601; 6,033,860; 6,090,555; 6,136,269;6,022,963; 6,083,697; 6,291,183; 6,309,831; 6,416,949; 6,428,752 and6,482,591, each of which is hereby incorporated by reference. A spottedmicroarray can also be used in a method according to an embodiment ofthe invention. An exemplary spotted microarray is a CodeLink™ Arrayavailable from Amersham Biosciences. Another microarray that is usefulis one that is manufactured using inkjet printing methods such asSurePrint™ Technology available from Agilent Technologies.

The systems and methods set forth herein can be used to detect thepresence of a particular target molecule in a sample contacted with themicroarray. This can be determined, for example, based on binding of alabeled target analyte to a particular probe of the microarray or due toa target-dependent modification of a particular probe to incorporate,remove, or alter a label at the probe location. Any one of severalassays can be used to identify or characterize targets using amicroarray as described, for example, in U.S. Patent ApplicationPublication Nos. 2003/0108867; 2003/0108900; 2003/0170684; 2003/0207295;or 2005/0181394, each of which is hereby incorporated by reference.

Exemplary labels that can be detected in accordance with embodiments ofthe invention, for example, when present on a microarray include, butare not limited to, a chromophore; luminophore; fluorophore; opticallyencoded nanoparticles; particles encoded with a diffraction-grating;electrochemiluminescent label such as Ru(bpy)³²⁺; or moiety that can bedetected based on an optical characteristic. Fluorophores that may beuseful include, for example, fluorescent lanthanide complexes, includingthose of Europium and Terbium, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, Cy3, Cy5, stilbene, Lucifer Yellow, CascadeBlue™, Texas Red, alexa dyes, phycoerythin, bodipy, and others known inthe art such as those described in Haugland, Molecular Probes Handbook,(Eugene, Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.),Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum PressNew York (1999), or WO 98/59066, each of which is hereby incorporated byreference.

In particular embodiments, the optical system can be configured for TimeDelay Integration (TDI) for example in line scanning embodiments asdescribed, for example, in U.S. Pat. No. 7,329,860, of which thecomplete subject matter is incorporated herein by reference in itsentirety. By way of example, the optical assembly may have a 0.75 NAlens and a focus accuracy of +/−125 to 500 nm. The resolution can be 50to 100 nm. The system may be able to obtain 1,000-10,000measurements/second unfiltered.

Although embodiments are exemplified with regard to detection of samplesthat includes biological or chemical substances supported by an opticalsubstrate, it will be understood that other samples can be analyzed,examined, or imaged by the embodiments described herein. Other exemplarysamples include, but are not limited to, biological specimens such ascells or tissues, electronic chips such as those used in computerprocessors, or the like. Examples of some of the applications includemicroscopy, satellite scanners, high-resolution reprographics,fluorescent image acquisition, analyzing and sequencing of nucleicacids, DNA sequencing, sequencing-by-synthesis, imaging of microarrays,imaging of holographically encoded microparticles and the like.

In other embodiments, the optical systems may be configured to inspectan object to determine certain features or structures of the object. Forexample, the optical systems may be used to inspect a surface of theobject, (e.g., semiconductor chip, silicon wafer) to determine whetherthere are any deviations or defects on the surface.

FIG. 1 illustrates a block diagram of an optical system 100 formed inaccordance with one embodiment. By way of example only, the opticalsystem 100 may be a sampler imager that images a sample of interest foranalysis. In other embodiments, the optical system 100 may be aprofilometer that determines a surface profile (e.g., topography) of anobject. Furthermore, various other types of optical systems may use themechanisms and systems described herein. In the illustrated embodiment,the optical system 100 includes an optical assembly 106, an objectholder 102 for supporting an object 110 near a focal plane FP of theoptical assembly 106, and a stage controller 115 that is configured tomove the object holder 102 in a lateral direction (along an X-axisand/or a Y-axis that extend into the page) or in a vertical/elevationaldirection along a Z-axis. The optical system 100 may also include asystem controller or computing system 120 that is operatively coupled tothe optical assembly 106, the stage controller 115, and/or the objectholder 102.

In particular embodiments, the optical system 100 is a sample imagerconfigured to image samples. Although not shown, a sample imager mayinclude other sub-systems or devices for performing various assayprotocols. By way of example only, the sample may include a flow cellhaving flow channels. The sample imager may include a fluid controlsystem that includes liquid reservoirs that are fluidicly coupled to theflow channels through a fluidic network. The sample imager may alsoinclude a temperature control system that may have a heater/coolerconfigured to regulate a temperature of the sample and/or the fluid thatflows through the sample. The temperature control system may includesensors that detect a temperature of the fluids.

As shown, the optical assembly 106 is configured to direct input lightto an object 110 and receive and direct output light to one or moredetectors. The output light may be input light that was at least one ofreflected and refracted by the object 110 and/or the output light may belight emitted from the object 110. To direct the input light, theoptical assembly 106 may include at least one reference light source 112and at least one excitation light source 114 that direct light, such aslight beams having predetermined wavelengths, through one or moreoptical components of the optical assembly 106. The optical assembly 106may include various optical components, including a conjugate lens 118,for directing the input light toward the object 110 and directing theoutput light toward the detector(s).

In the exemplary embodiment, the reference light source 112 may be usedby a distance measuring system or a focus-control system (or focusingmechanism) of the optical system 100 and the excitation light source 114may be used to excite the biological or chemical substances of theobject 110 when the object 110 includes a biological or chemical sample.The excitation light source 114 may be arranged to illuminate a bottomsurface of the object 110, such as in TIRF imaging, or may be arrangedto illuminate a top surface of the object 110, such as inepi-fluorescent imaging. As shown in FIG. 1, the conjugate lens 118directs the input light to a focal region 122 lying within the focalplane FP. The lens 118 has an optical axis 124 and is positioned aworking distance WD₁ away from the object 110 measured along the opticalaxis 124. The stage controller 115 may move the object 110 in theZ-direction to adjust the working distance WD₁ so that, for example, aportion of the object 110 is within the focal region 122.

To determine whether the object 110 is in focus (i.e., sufficientlywithin the focal region 122 or the focal plane FP), the optical assembly106 is configured to direct at least one pair of light beams to thefocal region 122 where the object 110 is approximately located. Theobject 110 reflects the light beams. More specifically, an exteriorsurface of the object 110 or an interface within the object 110 reflectsthe light beams. The reflected light beams then return to and propagatethrough the lens 118. As shown, each light beam has an optical path thatincludes a portion that has not yet been reflected by the object 110 anda portion that has been reflected by the object 110. The portions of theoptical paths prior to reflection are designated as incident light beams130A and 132A and are indicated with arrows pointing toward the object110. The portions of the optical paths that have been reflected by theobject 110 are designated as reflected light beams 130B and 132B and areindicated with arrows pointing away from the object 110. Forillustrative purposes, the light beams 130A, 130B, 132A, and 132B areshown as having different optical paths within the lens 118 and near theobject 110. However, in the exemplary embodiment, the light beams 130Aand 132B propagate in opposite directions and are configured to have thesame or substantially overlapping optical paths within the lens 118 andnear the object 110, and the light beams 130B and 132A propagate inopposite directions and are configured to have the same or substantiallyoverlapping optical paths within the lens 118 and near the object 110.

In the embodiment shown in FIG. 1, light beams 130A, 130B, 132A, and132B pass through the same lens that is used for imaging. In analternative embodiment, the light beams used for distance measurement orfocus determination can pass through a different lens that is not usedfor imaging. In this alternative embodiment, the lens 118 is dedicatedto passing beams 130A, 130B, 132A, and 132B for distance measurement orfocus determination, and a separate lens (not shown) is used for imagingthe object 110. Similarly, it will be understood that the systems andmethods set forth herein for focus determination and distancemeasurement can occur using a common objective lens that is shared withthe imaging optics or, alternatively, the objective lenses exemplifiedherein can be dedicated to focus determination or distance measurement.

The reflected light beams 130B and 132B propagate through the lens 118and may, optionally, be further directed by other optical components ofthe optical assembly 106. As shown, the reflected light beams 130B and132B are detected by at least one focus detector 144. In the illustratedembodiment, both reflected light beams 130B and 132B are detected by asingle focus detector 144. The reflected light beams may be used todetermine relative separation RS₁. For example, the relative separationRS₁ may be determined by the distance separating the beam spots from theimpinging reflected light beams 130B and 132B on the focus detector 144(i.e., a separation distance). The relative separation RS₁ may be usedto determine a degree-of-focus of the optical system 100 with respect tothe object 110. However, in alternative embodiments, each reflectedlight beam 130B and 132B may be detected by a separate correspondingfocus detector 144 and the relative separation RS₁ may be determinedbased upon a location of the beam spots on the corresponding focusdetectors 144.

If the object 110 is not within a sufficient degree-of-focus, thecomputing system 120 may operate the stage controller 115 to move theobject holder 102 to a desired position. Alternatively or in addition tomoving the object holder 102, the optical assembly 106 may be moved inthe Z-direction and/or along the XY plane. For example, the object 110may be relatively moved a distance AZ₁ toward the focal plane FP if theobject 110 is located above the focal plane FP (or focal region 122), orthe object 110 may be relatively moved a distance AZ₂ toward the focalplane FP if the object 110 is located below the focal plane FP (or focalregion 122). In some embodiments, the optical system 100 may substitutethe lens 118 with another lens 118 or other optical components to movethe focal region 122 of the optical assembly 106.

The example set forth above and in FIG. 1 has been presented withrespect to a system for controlling focus or for determiningdegree-of-focus. The system is also useful for determining the workingdistance WD₁ between the object 110 and the lens 118. In suchembodiments, the focus detector 144 can function as a working distancedetector and the distance separating the beam spots on the workingdistance detector can be used to determine the working distance betweenthe object 110 and the lens 118. For ease of description, variousembodiments of the systems and methods are exemplified herein withregard to controlling focus or determining degree-of-focus. It will beunderstood that the systems and methods can also be used to determinethe working distance between an object and a lens. Likewise, the systemsand methods may also be used to determine a surface profile of anobject.

In the exemplary embodiment, during operation, the excitation lightsource 114 directs input light (not shown) onto the object 110 to excitefluorescently-labeled biological or chemical substances. The labels ofthe biological or chemical substances provide light signals 140 (alsocalled light emissions) having predetermined wavelength(s). The lightsignals 140 are received by the lens 118 and then directed by otheroptical components of the optical assembly 106 to at least one objectdetector 142. Although the illustrated embodiment only shows one objectdetector 142, the object detector 142 may comprise multiple detectors.For example, the object detector 142 may include a first detectorconfigured to detect one or more wavelengths of light and a seconddetector configured to detect one or more different wavelengths oflight. The optical assembly 106 may include a lens/filter assembly thatdirects different light signals along different optical paths toward thecorresponding object detectors. Such optical systems are described infurther detail by PCT Application No. PCT/US07/07991, entitled “Systemand Devices for Sequence by Synthesis Analysis”, filed Mar. 30, 2007 andPCT Application No. PCT/US2008/077850, entitled “Fluorescence Excitationand Detection System and Method”, filed Sep. 26, 2008, both of which thecomplete subject matter are incorporated herein by reference in theirentirety.

The object detector 142 communicates object data relating to thedetected light signals 140 to the computing system 120. The computingsystem 120 may then record, process, analyze, and/or communicate thedata to other users or computing systems, including remote computingsystems through a communication line (e.g., Internet). By way ofexample, the object data may include imaging data that is processed togenerate an image(s) of the object 110. The images may then be analyzedby the computing system and/or a user of the optical system 100. Inother embodiments, the object data may not only include light emissionsfrom the biological or chemical substances, but may also include lightthat is at least one of reflected and refracted by the optical substrateor other components. For example, the light signals 140 may includelight that has been reflected by encoded microparticles, such as theholographically encoded optical identification elements described above.

In some embodiments, a single detector may provide both functions asdescribed above with respect to the object and focus detectors 142 and144. For example, a single detector may detect the reflected light beams130B and 132B and also the light signals 140.

The optical system 100 may include a user interface 125 that interactswith the user through the computing system 120. For example, the userinterface 125 may include a display (not shown) that shows and requestsinformation from a user and a user input device (not shown) to receiveuser inputs.

The computing system 120 may include, among other things, an objectanalysis module 150 and a focus-control module 152. The focus-controlmodule 152 is configured to receive focus data obtained by the focusdetector 144. The focus data may include signals representative of thebeam spots incident upon the focus detector 144. The data may beprocessed to determine relative separation (e.g., separation distancebetween the beam spots). A degree-of-focus of the optical system 100with respect to the object 110 may then be determined based upon therelative separation. In particular embodiments, the working distance WD₁between the object 110 and lens 118 can be determined. Likewise, theobject analysis module 150 may receive object data obtained by theobject detectors 142. The object analysis module may process or analyzethe object data to generate images of the object.

Furthermore, the computing system 120 may include any processor-based ormicroprocessor-based system, including systems using microcontrollers,reduced instruction set computers (RISC), application specificintegrated circuits (ASICs), field programmable gate array (FPGAs),logic circuits, and any other circuit or processor capable of executingfunctions described herein. The above examples are exemplary only, andare thus not intended to limit in any way the definition and/or meaningof the term system controller. In the exemplary embodiment, thecomputing system 120 executes a set of instructions that are stored inone or more storage elements, memories, or modules in order to at leastone of obtain and analyze object data. Storage elements may be in theform of information sources or physical memory elements within theoptical system 100.

The set of instructions may include various commands that instruct theoptical system 100 to perform specific protocols. For example, the setof instructions may include various commands for performing assays andimaging the object 110 or for determining a surface profile of theobject 110. The set of instructions may be in the form of a softwareprogram. As used herein, the terms “software” and “firmware” areinterchangeable, and include any computer program stored in memory forexecution by a computer, including RAM memory, ROM memory, EPROM memory,EEPROM memory, and non-volatile RAM (NVRAM) memory. The above memorytypes are exemplary only, and are thus not limiting as to the types ofmemory usable for storage of a computer program.

As described above, the excitation light source 114 generates anexcitation light that is directed onto the object 110. The excitationlight source 114 may generate one or more laser beams at one or morepredetermined excitation wavelengths. The light may be moved in a rasterpattern across portions of the object 110, such as groups in columns androws of the object 110. Alternatively, the excitation light mayilluminate one or more entire regions of the object 110 at one time andserially stop through the regions in a “step and shoot” scanningpattern. Line scanning can also be used as described, for example, inU.S. Pat. No. 7,329,860, of which the complete subject matter isincorporated herein by reference in its entirety. The object 110produces the light signals 140, which may include light emissionsgenerated in response to illumination of a label in the object 110and/or light that has been reflected or refracted by an opticalsubstrate of the object 110. Alternatively, the light signals 140 may begenerated, without illumination, based entirely on emission propertiesof a material within the object 110 (e.g., a radioactive orchemiluminescent component in the object).

The object and focus detectors 142 and 144 may be, for examplephotodiodes or cameras. In some embodiments herein, the detectors 142and 144 may comprise a camera that has a 1 mega pixel CCD-based opticalimaging system such as a 1002×1004 CCD camera with 8 μm pixels, which at20× magnification can optionally image an area of 0.4×0.4 mm per tileusing an excitation light that has a laser spot size of 0.5×0.5 mm(e.g., a square spot, or a circle of 0.5 mm diameter, or an ellipticalspot, etc.). Cameras can optionally have more or less than 1 millionpixels, for example a 4 mega pixel camera can be used. In manyembodiments, it is desired that the readout rate of the camera should beas fast as possible, for example the transfer rate can be 10 MHz orhigher, for example 20 or 30 MHz. More pixels generally mean that alarger area of surface, and therefore more sequencing reactions or otheroptically detectable events, can be imaged simultaneously for a singleexposure. In particular embodiments, the CCD camera/TIRE lasers maycollect about 6400 images to interrogate 1600 tiles (since images areoptionally done in 4 different colors per cycle using combinations offilters, dichroics and detectors as described herein). For a 1 Megapixel CCD, certain images optionally can contain between about 5,000 to50,000 randomly spaced unique nucleic acid clusters (i.e., images uponthe flow cell surface). At an imaging rate of 2 seconds per tile for thefour colors, and a density of 25000 clusters per tile, the systemsherein can optionally quantify about 45 million features per hour. At afaster imaging rate, and higher cluster density, the imaging rate can beimproved. For example, a readout rate of a 20 MHz camera, and a resolvedcluster every 20 pixels, the readout can be 1 million clusters persecond. A detector can be configured for Time Delay Integration (TDI)for example in line scanning embodiments as described, for example, inU.S. Pat. No. 7,329,860, of which the complete subject matter isincorporated herein by reference in its entirety. Other useful detectorsinclude, but are not limited, to an optical quadrant photodiodedetector, such as those having a 2×2 array of individual photodiodeactive areas fabricated on a single chip, examples of which areavailable from Pacific Silicon Sensor (Westlake Village, Calif.), or aposition sensitive detector such as those having a monolithic PINphotodiode with a uniform resistance in one or two dimensions, examplesof which are available from Hamamatsu Photonics, K.K., (Hamamatsu City,Japan).

FIGS. 2 and 3 illustrate perspective and plan views of an opticalassembly 202 formed in accordance with one embodiment. The opticalassembly 202 may be used with the optical system 100 (FIG. 1) or otheroptical systems. As shown, the optical assembly 202 includes an opticaltrain 240 of optical components 241-245 that direct light beams 230 and232 along an optical track or course between an object of interest (notshown) and a focus detector 250. In particular embodiments, the focusdetector can also be referred to as a distance detector. The series ofoptical components 241-245 of the optical train 240 include a dual-beamgenerator 241, a beam splitter 242, a conjugate lens 243, a beamcombiner 244, and a fold mirror 245.

The optical assembly 202 includes a reference light source 212 thatprovides a light beam 228 to the dual-beam generator 241. The referencelight source 212 may be, for example, a 660 nm laser. The dual-beamgenerator 241 provides a pair of parallel incident light beams 230A and232A and directs the incident light beams 230A and 232A toward the beamsplitter 242. In the illustrated embodiment, the dual-beam generator 241comprises a single body having opposite parallel surfaces 260 and 262(FIG. 3). The first surface 260 reflects a portion of the light beam 228that forms the incident light beam 230A and refracts a portion of thelight beam 228. The refracted portion of the light beam 228 is reflectedby the opposite second surface 262 toward the first surface 260, whichforms the incident light beam 232A.

The dual-beam generator 241 directs the parallel incident light beams230A and 232A toward the beam splitter 242. The beam splitter 242reflects the incident light beams 230A and 232A toward the conjugatelens 243. In the exemplary embodiment, the beam splitter 242 includes apair of reflectors (e.g., aluminized tabs) that are positioned toreflect the incident light beams 230A and 232A and the reflected lightbeams 230B and 232B. The beam splitter 242 is positioned to reflect theincident light beams 230A and 232A so that the incident light beams 230Aand 232A propagate parallel to an optical axis 252 of the lens 243. Theoptical axis 252 extends through a center of the lens 243 and intersectsa focal region 256 (FIG. 2). The lens 243 may be a near-infinityconjugated objective lens. In alternative embodiments, the incidentlight beams 230A and 232A propagate in a non-parallel manner withrespect to the optical axis 252. Also shown in FIG. 3, the incidentlight beams 230A and 232A may be equally spaced apart from the opticalaxis 252 as the incident light beams 230A and 232A propagate through thelens 243.

As described above with respect to the optical system 100, the incidentlight beams 230A and 232A converge toward the focal region 256 (FIG. 2)and are reflected by an object 268 (shown in FIG. 4) located proximateto the focal region 256 and return to and propagate through the lens 243as reflected light beams 230B and 232B. The reflected light beams 230Band 232B may propagate along a substantially equal or overlappingoptical path with respect to the incident light beams 232A and 230A,respectively, through the lens 243 and toward the dual-beam generator241. More specifically, the reflected light beam 230B propagates in anopposite direction along substantially the same optical path of theincident light beam 232A, and the reflected light beam 232B propagatesin an opposite direction along substantially the same optical path ofthe incident light beam 230A. The reflected light beams 230B and 232Bexit the lens 243 separated by a path spacing PS₂ that is substantiallyequal to a path spacing PS₁ that separates the incident light beams 230Aand 232A (shown in FIG. 3).

As shown in FIGS. 2 and 3, the reflected light beams 230B and 232B areincident upon and directed by the dual-beam generator 241 through arange limiter 254 toward the beam combiner 244. In the illustratedembodiment, the beam combiner 244 is configured to modify the pathspacing PS that separates the reflected light beams 230B and 232B. Thepath spacing PS at the beam combiner 244 may be re-scaled to besubstantially equal to a separation distance SD₁ of the reflected lightbeams 230B and 232B detected by the focus detector 250. The separationdistance SD₁ is a distance measured between the reflected light beams ata predetermined portion of the optical track, such as at the focusdetector 250. In particular embodiments, the separation distance SD₁ atthe focus detector 250 is less than the path spacing PS at the beamcombiner 244 so that only a single focus detector 250 may detect bothreflected light beams 230B and 232B. Furthermore, the beam combiner 244may substantially equalize the optical path lengths of the reflectedlight beams 230B and 232B.

The reflected light beams 230B and 232B propagate substantially parallelto each other between optical components after exiting the lens 243. Inthe illustrated embodiment, the reflected light beams 230B and 232Bpropagate substantially parallel to each other along the optical trackbetween the lens 243 and the focus detector 250. As used herein, twolight beams propagate “substantially parallel” to one another if the twolight beams are essentially co-planar and, if allowed to propagateinfinitely, would not intersect each other or converge/diverge withrespect to each other at a slow rate. For instance, two light beams aresubstantially parallel if an angle of intersection is less than 20° or,more particularly, less than 10° or even more particularly less than 1°.For instance, the reflected light beams 230B and 232B may propagatesubstantially parallel to each other between the beam splitter 242 andthe dual-beam generator 241; between the dual-beam generator 241 and thebeam combiner 244; between the beam combiner 244 and the fold mirror245; and between the fold mirror 245 and the focus detector 250.

The optical train 240 is configured to maintain a projectionrelationship (described further below) between the reflected light beams230B and 232B throughout the optical track so that a degree-of-focus maybe determined. By way of example, if the optical assembly 202 is infocus with the object, the reflected light beams 230B and 232B willpropagate parallel to each other between each optical component in theoptical train 240. If the optical assembly 202 is not in focus with theobject, the reflected light beams 230B and 232B are co-planar, butpropagate at slight angles with respect to each other. For example, thereflected light beams 230B and 232B may diverge from each other orconverge toward each other as the reflected light beams 230B and 232Btravel along the optical track to the focus detector 250.

To this end, each optical component 241-245 may have one or moresurfaces that are shaped and oriented to at least one of reflect andrefract the reflected light beams 230B and 232B so that the reflectedlight beams 230B and 232B maintain the projection relationship betweenthe reflected light beams 230B and 232B. For example, the opticalcomponents 242 and 245 have a planar surface that reflects both of theincident light beams 230B and 232B. The optical components 241 and 244may also have parallel surfaces that each reflects one of the incidentlight beams 230B and 232B. Accordingly, if the reflected light beams230B and 232B are parallel, the reflected light beams 230B and 232B willremain parallel to each other after exiting each optical component. Ifthe reflected light beams 230B and 232B are converging or divergingtoward each other at certain rate, the reflected light beams 230B and232B will be converging or diverging toward each other at the same rateafter exiting each optical component. Accordingly, the opticalcomponents along the optical track may include a planar surface thatreflects at least one of the reflected light beams or a pair of parallelsurfaces where each surface reflects a corresponding one of thereflected light beams.

An optical system can include one or more optical assemblies fordetermination of a working distance or focus. For example, an opticalsystem can include two optical assemblies of the type shown in FIGS. 2and 3 to allow focus to be determined at two different positions on anobject or to provide for determination of the working distance betweenthe optical system and the object at two different positions. Forembodiments, in which more than one optical assembly is present, theoptical assemblies can be discrete and separate or the opticalassemblies can share optical components. The optical assemblies canshare optical components such as reference light source 212, focusdetector 250, fold mirror 245, beam combiner 244, dual-beam generator241, beam splitter 242, epi-fluorescent (EPI) input reflector 280 andrange limiter 254. Optical components can be shared by placing a beamsplitter upstream of the shared components in the optical train.Although exemplified for the optical assembly shown in FIGS. 2 and 3,one or more versions of other optical assemblies that are exemplifiedherein can be present in a particular optical system. Furthermore, aparticular optical system can include various combinations of theoptical assemblies set forth herein.

As shown in FIG. 3, the reflected light beams 230B and 232B areultimately incident upon a detector surface 264 of the focus detector250 at corresponding beam spots. The beam spots are spaced apart by aseparation distance SD₁. The separation distance SD₁ indicates whetherthe optical assembly 202 has a sufficient degree-of-focus with respectto the object. The separation distance SD₁ on the detector surface 264also indicates a working distance between lens 243 and the object beingimaged.

In other embodiments, the optical components 241-245 may be substitutedwith alternative optical components that perform substantially the samefunction as described above. For example, the beam splitter 242 may bereplaced with a prism that directs the incident light beams 230A and232A through the lens 243 parallel to the optical axis 252. The beamcombiner 244 may not be used or may be replaced with an optical flatthat does not affect the path spacing of the reflected light beams.Furthermore, the optical components 241-245 may have different sizes andshapes and be arranged in different configurations or orientations asdesired. For example, the optical train 240 of the optical assembly 202may be configured for a compact design.

Furthermore, in alternative embodiments, the parallel light beams may beprovided without the dual-beam generator 241. For example, a referencelight source 212 may include a pair of light sources that are configuredto provide parallel incident light beams. In alternative embodiments,the focus detector 250 may include two focus detectors arrangedside-by-side in fixed, known positions with respect to each other. Eachfocus detector may detect a separate reflected light beam. Relativeseparation between the reflected light beams may be determined based onthe positions of the beam spots with the respective focus detectors andthe relative position of the focus detectors with respect to each other.

Although not illustrated in FIGS. 2 and 3, the optical assembly 202 mayalso be configured to facilitate collecting output light that isprojected from the object 268. For example, the optical assembly 202 mayinclude an epi-fluorescent (EPI) input reflector 280 that is positionedto reflect incident light that is provided by an excitation light source(not shown). The light may be directed toward the beam splitter 242 thatreflects at least a portion of the excitation light and directs thelight along the optical axis 252 through the lens 243. The lens 243directs the light onto the object 268, which may provide the outputlight. The lens 243 then receives the output light (e.g., lightemissions) from the object 268 and direct the output light back towardthe beam splitter 242. The beam splitter 242 may permit a portion of theoutput light to propagate therethrough along the optical axis. Theoutput light may then be detected by an object detector (not show).

As shown in FIG. 3, the EPI input reflector 280 includes two passages282 and 284 that allow the light beams 230 and 232 to propagatetherethrough without being affected by the input reflector 280.Accordingly, the beam splitter 242 may reflect the incident andreflected light beams 230A, 230B, 232A, and 232B and may also reflectthe excitation light.

FIGS. 4-9 show different projection relationships between reflectedlight beams 230B and 232B and corresponding beam spots 270 and 272 onthe detector surface 264. As discussed above, the projectionrelationship between the reflected light beams is based upon where theobject is located in relation to the focal region. When the object ismoved with respect to the focal region, the projection relationshipbetween the reflected light beams changes and, consequently, therelative separation between the reflected light beams also changes.FIGS. 4-9 illustrate how a separation distance SD measured between beamspots may change as the projection relationship between the reflectedlight beams change. However, the separation distance SD is just onemanner of determining relative separation between the reflected lightbeams. Accordingly, those skilled in the art understand that FIGS. 4-9illustrate only one manner of determining the relative separation andthat other manners for determining relative separation or the projectionrelationship are possible.

FIGS. 4 and 5 show a projection relationship between reflected lightbeams 230B and 232B when the optical assembly 202 (FIG. 2) is in focuswith respect to an object 268. As shown, the incident light beams 230Aand 232A propagate through the lens 243 parallel to each other andspaced apart by a path spacing PS₁. In the illustrated embodiment, theincident light beams 230A and 232A propagate parallel to the opticalaxis 252 of the lens 243 and are equidistant from the optical axis 252.In alternative embodiments, the incident light beams 230A and 232A maypropagate in a non-parallel manner with respect to the optical axis 252and have different spacings therefrom. In a particular alternativeembodiment, one of the incident light beams 230A or 232A coincides withthe optical axis 252 of the lens 243 and the other is spaced apart fromthe optical axis 252.

The incident light beams 230A and 232A are directed by the lens 243 toconverge toward the focal region 256. In such embodiments where theincident light beams are non-parallel to the optical axis, the focalregion may have a different location than the location shown in FIG. 4.The incident light beams 230A and 232A are reflected by the object 268and form the reflected light beams 230B and 232B. The reflected lightbeams 230B and 232B return to and propagate through the lens 243 andparallel to the optical axis 252. The reflected light beams 230B and232B exit the lens 243 parallel to each other and spaced apart by a pathspacing PS₂. When the optical assembly 202 is in focus, the pathspacings PS₁ and PS₂ are equal.

Accordingly, when the optical assembly 202 is in focus, the projectionrelationship of the reflected light beams 230B and 232B exiting the lens243 includes two parallel light beams. The optical train 240 isconfigured to maintain the parallel projection relationship. Forexample, when the optical assembly 202 is in focus, the reflected lightbeams 230B and 232B are parallel to each other when exiting thedual-beam generator 241, when exiting the beam combiner 244, and whenreflected by the fold mirror 245. Although the projection relationshipis maintained, the path spacing PS₂ may be re-scaled by a beam combiner.

As shown in FIG. 5, the reflected light beams 230B and 232B of FIG. 4are incident upon the detector surface 264 and form the beam spots 270and 272. When the optical assembly 202 is in focus, the beam spots 270and 272 have a separation distance SD₂. The separation distance SD₂ canbe based upon (or a function of) dimensions of the beam combiner 244 andan angle of incidence with respect to the parallel surfaces of the beamcombiner 244 and the impinging reflected light beams 230B and 232B. Theseparation distance SD₂ is also based upon the projection relationshipof the reflected light beams 230B and 232B exiting the lens 243. Asshown in FIG. 5, the detector surface 264 has a center point or region266. If all of the optical components 241-245 (FIG. 2) of the opticaltrain 240 are in respective desired positions, the beam spots 270 and272 may be equally spaced apart from the center region 266 along anX-axis and vertically centered within the detector surface 264. Alsoshown, the beam spots 270 and 272 may have a select morphology that iscorrelated with the optical assembly 202 being in focus. For example,the beam spots 270 and 272 may have an airy radius that correlates tothe optical assembly 202 being in focus.

FIGS. 6 and 7 show a projection relationship between the reflected lightbeams 230B and 232B when the optical assembly 202 (FIG. 2) is belowfocus. As described above, the incident light beams 230A and 232Apropagate through the lens 243 parallel to each other and spaced apartby the path spacing PS₁. The incident light beams 230A and 232Aintersect each other at the focal region 256 and are then reflected bythe object 268 to form the reflected light beams 230B and 232B. However,as shown in FIG. 6, when the reflected light beams 230B and 232B exitthe lens 243, the reflected light beams 230B and 232B are slightlyconverging toward the optical axis 252 and each other. Also shown, thepath spacing PS₂ is greater than the path spacing PS₁.

Accordingly, when the object 268 is located below the focal region 256,the projection relationship of the reflected light beams 230B and 232Bincludes two light beams that converge toward each other. Similar toabove, the optical train 240 is configured to maintain the convergingprojection relationship. For example, the reflected light beams 230B and232B are converging toward each other when exiting the dual-beamgenerator 241, when exiting the beam combiner 244, and when reflected bythe fold mirror 245.

As shown in FIG. 7, when the object 268 is located below the focalregion 256, the beam spots 270 and 272 have a separation distance SD₃that is less than the separation distance SD₂ (FIG. 5). The separationdistance SD₃ is less because the reflected light beams 130B and 132Bconverge toward each other throughout the optical track between the lens243 and the focus detector 250. Also shown in FIG. 7, the beam spots 270and 272 may have a select morphology that is correlated with the beamspots 270 and 272. The morphology of the beam spots 270 and 272 when theobject 268 is located below the focal region 256 is different than themorphology of the beam spots 270 and 272 when the object 268 is infocus. The beam spots 270 and 272 may have a different airy radius thatcorrelates to the object being below the focal region 256.

FIGS. 8 and 9 show a projection relationship between the reflected lightbeams 230B and 232B when the optical assembly 202 (FIG. 2) is abovefocus. As described above, the incident light beams 230A and 232Apropagate through the lens 243 parallel to each other and spaced apartby the path spacing PS₁. Before the incident light beams 230A and 232Areach the focal region 256, the incident light beams 230A and 232A arereflected by the object 268 to form the reflected light beams 230B and232B. However, as shown in FIG. 8, when the reflected light beams 230Band 232B exit the lens 243, the reflected light beams 230B and 232Bdiverge away from the optical axis 252 and away from each other. Alsoshown, the path spacing PS₂ is less than the path spacing PS₁.

Accordingly, when the object 268 is located above the focal region 256,the projection relationship of the reflected light beams 230B and 232Bincludes two light beams that diverge away from each other. The opticaltrain 240 is configured to maintain the diverging projectionrelationship. For example, the reflected light beams 230B and 232B arediverging away from each other when exiting the dual-beam generator 241,when exiting the beam combiner 244, and when reflected by the foldmirror 245.

As shown in FIG. 9, when the object 268 is located above the focalregion 256, the beam spots 270 and 272 have a separation distance SD₄that is greater than the separation distance SD₂. The separationdistance SD₄ is greater because the reflected light beams 130B and 132Bdiverge from each other throughout the optical track between the lens243 and the focus detector 250. Also shown in FIG. 9, the beam spots 270and 272 may have a select morphology that is correlated with the beamspots 270 and 272. The morphology of the beam spots 270 and 272 when theobject 268 is located above the focal region 256 is different than themorphology of the beam spots 270 and 272 when the object 268 is in focusor below the focal region 256. Likewise, the beam spots 270 and 272 mayhave a different airy radius that correlates to the object being belowthe focal region 256.

As described above, if the object 268 is below the focal region 256, theseparation distance SD₃ is less than the separation distance SD₂ inwhich the object 268 is within the focal region 256. If the object 268is above the focal region 256, the separation distance SD₄ is greaterthan the separation distance SD₂. As such, the optical assembly 202 notonly determines that the object 268 is not located within the focalregion 256, but may also determine a direction to move the object 268with respect to the lens 243. Furthermore, a value of the separationdistance SD₃ may be used to determine how far to move the object 268with respect to the lens 243. As set forth elsewhere herein, ameasurement of separation distance on a detector can be used todetermine the working distance between the lens and an object that isbeing detected through the lens. Furthermore, the separation distance onthe detector may be used to determine a profile of an object surface.

Accordingly, relative separation (e.g., a separation distance) is afunction of the projection relationship (i.e., what rate the reflectedlight beams 230B and 232B are diverging or converging) and a length ofthe optical track measured from the lens 243 to the focus detector 250.As the optical track between the lens 243 and the focus detector 250increases in length, the separation distance decreases or increases ifthe object is not in focus. As such, the length of the optical track maybe configured to facilitate distinguishing the separation distances SD₃and SD₄. For example, the optical track may be configured so thatconverging reflected light beams do not cross each other and/orconfigured so that diverging light beams do not exceed a predeterminedrelative separation between each other. To this end, the optical trackbetween optical components of the optical train 240 may be lengthened orshortened as desired.

Furthermore, additional optical components, such as a beam foldingdevice, may be added to increase the length of the optical path. A beamfolding device or other device for increasing the optical path lengthcan act as an amplifier since the increase in path length for two beamsthat deviate from parallel will increase the magnitude of the deviationas perceived on the surface of a detector that intersects the two beams(i.e. increased path length will increase the separation distance forspots generated from diverging beams and will decrease the separationdistance for spots generated from converging beams).

In particular embodiments, the computing system that receives the focusdata from the focus detector 250 only identifies a centroid of each beamspot to determine the separation distance SD. However, the computingsystem may also analyze a morphology of each beam spot. As shown above,the beam spots 270 and 272 may have different airy radiuses (or disks)based upon the degree-of-focus of the optical assembly 202. The airyradiuses may be analyzed in addition to the separation distance SD todetermine a degree-of-focus of the optical assembly 202.

FIG. 10 illustrates alternative embodiments for determining relativeseparation between reflected light beams 902 and 904. As shown in FIG.10, the reflected light beams 902 and 904 exit the lens 906 having adiverging projection relationship. However, the projection relationshipmay also be parallel or converging. The reflected light beams 902 and904 may be redirected along an optical track by an optical train 908(generally indicated by a dashed box).

FIG. 10 illustrates various embodiments in which spot detectors haveknown spatial relationships with respect to each other and detectcorresponding beam spots. The spot detectors may be, for example, focusdetectors or working distance detectors as discussed above. In a firstembodiment, the reflected light beam 902 may have an optical path 912and is incident upon a spot detector 916. The reflected light beam 904may have an optical path 914 and is incident upon a spot detector 918.(Optical components 933 and 935 are provided as dashed boxes to indicatethat the optical components are optionally present, for example, beingnot present in a first embodiment of FIG. 10.) The spot detectors 916and 918 have a known spatial relationship with respect to each other.For example, the spot detectors 916 and 918 may be oriented to face acommon direction and be spaced apart a distance D₁. Each of the spotdetectors 916 and 918 may detect a corresponding beam spot 922 and 924from the reflected light beams 902 and 904, respectively.

As described above, when an object is moved with respect to the focalregion, the projection relationship between the reflected light beamchanges. When the projection relationship changes, locations of the beamspots on the detector surfaces move in a predetermined manner. Thechange in location by each beam spot may be used to determine adegree-of-focus of the object, a working distance from the lens to theobject, or a surface profile of the object. More specifically, thechange in spot location for each reflected beam may be used to determinerelative separation between the reflected light beams 902 and 904. Inthe first embodiment, the beam spots 922 and 924 move in a similarmanner as described with respect to the beam spots 270 and 272 in FIGS.4-9.

In a second embodiment shown in FIG. 10, the reflected light beam 902may have an optical path 932 where the beam is reflected, separately, byan optical component 933 and is incident upon a spot detector 936. Thereflected light beam 904 may have an optical path 934 in which the beamis reflected, separately, by an optical component 935 and is incidentupon a spot detector 938. The spot detectors 936 and 938 have a knownspatial relationship with respect to each other. The spot detectors 936and 938 directly face each other. As shown, each of the spot detectors936 and 938 may detect a corresponding beam spot 942 and 944 from thereflected light beams 902 and 904, respectively. In the secondembodiment, the beam spots 942 and 944 move in a common direction whenthe projection relationship of the reflected light beams 902 and 904changes. Relative separation may be determined as a function of thedistance and direction moved by the beam spots 942 and 944.

The second embodiment may be used in optical systems where, for example,system space is limited or restricted. Furthermore, the secondembodiment may be used when it is desirable to equalize optical pathlengths of the reflected light beams 930 and 932. For example, theoptical components 933 and 935 may be located different distances D₂ andD₃ away from the corresponding spot detectors 936 and 938 to equalizethe optical path lengths. In alternative embodiments, optical systemsmay have different configurations of spot detectors as shown in FIG. 10.For example, in one embodiment, the optical system may have spotdetectors 918 and 936. Accordingly, optical systems may determinerelative separation based not only on spot locations on one or more spotdetectors, but also upon the spatial relationships between the spotdetectors.

FIGS. 11-14 illustrate an effect on reflected light beams by two opticalcomponents when one of the optical components is mispositioned.Throughout the lifetime of an optical system, various optical componentsused by the focusing mechanism may shift, rotate, or otherwise be movedfrom a desired or preset position. As shown in FIG. 11, opticalcomponents 341 and 342 are positioned relative to each other and areconfigured to facilitate redirecting reflected light beams toward adetector surface 364 (shown in FIG. 12). The optical component 341 isshown in both a desired position (indicated by solid lines) and in amisoriented position (indicated by dashed lines) where the opticalcomponent 341 is rotated about an axis 390. The reflected light beamsare shown in a desired optical path (indicated by solid lines) and in anoptical path (indicated by dashed lines) in which the optical component341 has been slightly rotated.

FIG. 12 illustrates beam spots 370 and 372 on a detector surface 364that are provided by the different sets of reflected light beams shownin FIG. 11. Beam spots 370A and 372A illustrate a relative location ofthe beam spots when the optical components 341 and 342 are properlypositioned. Beam spots 370B and 372B illustrate a relative location ofthe beam spots when the optical components 341 and 342 are not properlypositioned. The beam spots 370A and 372A have a separation distance SD₅,and the beam spots 370B and 372B have a separation distance SD₆. Asshown, the separation distances SD₅ and SD₆ are substantially equal. Theseparation distances SD₅ and SD₆ are substantially equal because eachreflected light beam is similarly affected by the optical component 341.As such, the separation distance SD may be maintained even when one ofthe optical components is mispositioned.

However, as shown in FIG. 12, the pair of beam spots 370B and 372B havebeen shifted a lateral offset 392 due to the movement of the opticalcomponent 341 from the desired position. The computing system can beconfigured to determine focus based on relative separation (e.g.,separation distance) between beam spots 370B and 372B, independent ofthe offset. The computing system may determine that the beam spots 370and 372 have been shifted together from desired locations. For example,the computing system may determine a common drift by the beam spots 370and 372 in which the beam spots have moved in a common direction anddistance away from an original or a desired location. Such informationcan be used as a system diagnostic, for example, alerting the computingsystem that at least one of the optical components has moved from thedesired position(s).

As shown in FIG. 13, the optical components 341 and 342 are positionedrelative to each other and are configured to facilitate redirectingreflected light beams toward a detector surface 364 (shown in FIG. 14).The optical component 342 is shown in a misoriented position where theoptical component 342 has been rotated about an axis 394 from theposition shown in FIG. 10. As shown, the reflected light beams arereflected by the optical component 342 such that the light beams projectat an angle away from the plane of the page.

FIG. 14 illustrates the beam spots 370 and 372 on the detector surface364 that are provided by the reflected light beams shown in FIG. 13.Beam spots 370A and 372A illustrate a relative location of the beamspots when the optical components 341 and 342 are properly positioned.Beam spots 370B and 372B illustrate a relative location of the beamspots when the optical components 341 and 342 are not properlypositioned. The beam spots 370A and 372A have a separation distance SD₇,and the beam spots 370B and 372B have a separation distance SD₈. Asshown, the separation distances SD₇ and SD₈ are substantially equal. Theseparation distances SD₇ and SD₈ are substantially equal because eachreflected light beam is similarly affected by the optical component 342.Similarly, the pair of beam spots 370B and 372B has been shifted by avertical offset 396 due to the movement of the optical component 341from the desired position.

The computing system may determine that the beam spots 370 and 372 havebeen shifted together from desired locations. For example, the computingsystem may determine a common drift by the beam spots 370 and 372 inwhich the beam spots have moved in a common direction and distance awayfrom an original or a desired location. Such information can be used forsystem diagnostics to identify that components are misaligned and insome embodiments to indicate the nature or type of misalignment.

As illustrated by the examples shown in FIGS. 11 through 14, anadvantage of particular embodiments of the invention is that focus canbe determined independent of misalignment of optical components. Incontrast, many other focusing systems that rely on location of a beamspot relative to a fixed location can be subject to error due tomisalignment of optical components. This can in turn require anundesirable level of attention to system calibration than necessary forembodiments of the present invention. Similarly the working distancebetween an objective lens and an object can be determined independent ofsuch misalignment of optical components.

In some embodiments, one or more of the optical components in theoptical train may be selectively moved. For example, if the pair of beamspots 370 and 372 were to drift so much that one or more of the beamspots is undetectable, the optical system could selectively move atleast one of the optical components 341 to 342 to move the pair of beamspots to an acceptable position. The optical components 341 and 342could be at least one of rotated or shifted to a different position.

Furthermore, in alternative embodiments, one or more of the opticalcomponents could be selectively moved to redirect the reflected lightbeams to a different detector. For example, if the reflected light beamsrequire a different type of detection or a different sized detectorsurface due to a change in conjugate lenses, one or more of the opticalcomponents could be moved to change the optical track and direct thereflected light beams to a different detector.

With reference to the illustrated embodiment shown in FIGS. 4-9, theincident light beams 230A and 232A are equally off-center from theoptical axis 252. However, in alternative embodiments, the incidentlight beams 230A and 232A may be located at different positions. Forexample, the incident light beam 230A may be located a greater distanceor spacing from the optical axis 252 than the incident light beam 232A.In such embodiments, the beam spots 270 and 272 may still be used todetermine separation distance. However, due to the different spacingsfrom the optical axis 252, the beam spots 270 and 272 may have positionchanges of different magnitude on the detector surface 264. For example,the beam spot 270 may move a larger distance on the detector surface 264than the distance moved by beam spot 272 on the detector surface 264.Nevertheless, the expected difference in the magnitude of change can betaken into account in order to accurately determine degree of focus orworking distance for the optical system based on the relative separationbetween beam spot 270 and beam spot 272.

In other embodiments, one of the incident light beams may coincide withthe optical axis 252 while the other light beam is spaced apart from andpropagates along the optical axis 252. In such embodiments, the incidentlight beam that is spaced apart from the optical axis will move when theobject is moved out of focus as described above. However, the beam spotcorresponding to the incident light beam that coincides with the opticalaxis 252 will not move on the detector surface 264 when the object ismoved out of focus. Nonetheless, changes in the relative separationbetween the beam spots can be used to determine degree of focus orworking distance for the optical system. Furthermore, changes in theposition of the optical components (e.g., rotation, drift) or otheradverse affects can cause both beam spots to move. In this embodiment,movement of the beam that coincides with the optical axis will beindicative of the altered alignment of the optical components and willbe distinguishable from a change in focus or a change in workingdistance between the lens and object being viewed. The distinction canbe made because a change in focus or working distance would not causemovement of the beam that coincides with the optical axis absent achange in alignment of components. Thus, use of a beam that coincideswith the optical axis can be advantageous in providing diagnosticinformation about alignment of the optical system in addition toproviding information about the degree of focus or working distancebetween an objective lens and an object being viewed.

FIG. 15 is a side view of a flow cell 400 that may be used in variousembodiments. When the object includes a flow cell or other opticalsubstrates having multiple layers of different refractive index, theincident light beams may be reflected at multiple points within the flowcell. For example, the flow cell 400 includes a cover slip or top layer402 having opposite surfaces 404 and 406 and a bottom layer 408 havingopposite surfaces 410 and 412. As shown, the top and bottom layers 402and 408 have a flow channel 414 of the flow cell 400 extendingtherebetween. The flow channel 414 may include a fluid F flowingtherethrough. When incident light beams 430A and 432A are directedtoward a focal region 456 within or along the flow cell 400, theincident light beams 430A and 432A may be reflected by the flow cell 400at multiple points P along an optical axis 452 of the lens (not shown).For example, if the optical assembly is configured to scan the surface410 of the bottom layer 408, the incident light beams 430A and 432A arereflected at reflection points P₁-P₄ prior to reaching the focal region456. Such reflection provides unwanted reflected light beams (indicatedby dashed lines). The unwanted reflected light beams may be detected bya focus detector (not shown).

Accordingly, in some embodiments, an optical assembly may include arange limiter, such as the range limiter 254 shown in FIGS. 2 and 3.Range limiters include slits or openings that filter or remove theunwanted reflected light beams from the output light so that the focusdetector does not misinterpret the light signals that impinge on thesurface of the focus detector. The ranger limiter 254 may be configuredto filter out reflected light beams that are not within, for example,+/−20 um from the focal region 456. The range limiter includes oneopening for each reflected light beam, such as the reflected light beams230B and 232B shown in FIGS. 2 and 3. Furthermore, reflected light beamsthat diverge or converge at an excessive angle may also be filtered outby the ranger limiter.

FIGS. 16A and 16B illustrate alternative optical assemblies 850 and 880.In various embodiments, optical systems described herein may use aplurality of pairs of incident light beams. For example, the opticalassembly 850 includes a conjugate lens 854 and optical components 860and 862 to deliver and receive light beams as described herein tofacilitate imaging/scanning/profiling an object 852. The opticalcomponent 860 is configured to direct a first pair of parallel incidentlights beams 870 and 872 parallel to an optical axis 875 of the lens 854such that the first pair of incident light beams 870 and 872 aredirected toward a focal region 856A located on the optical axis 875. Theoptical component 862 is configured to direct a second pair of parallelincident lights beams 874 and 876 to the lens 854 at a non-orthogonalangle with respect to the optical axis 875 such that the second pair ofincident light beams 874 and 876 are directed toward a focal region 856Blocated at a different location on the focal plane.

FIG. 16B illustrates the optical assembly 880, which includes aconjugate lens 882 and a common optical component 884. The opticalcomponent 884 is configured to direct multiple pairs of incident lightbeams to a conjugate lens 886. In the illustrated embodiment, theoptical component 884 is a beam generator such as those described above.Although not shown, the optical component 884 may receive separate lightbeams from separate light sources in which the light beams are incidentupon the optical component 884 at different angles. As such, the opticalcomponent 884 may generate first and second pairs 890 and 892 ofincident light beams that are non-parallel with respect to each other.As described above with respect to FIG. 16A, the first and second pairs890 and 892 of incident light beams are directed toward separate focalregions 888A and 888B located on a common focal plane.

The optical assemblies 850 and 880 may be used in various opticalsystems for various purposes. In both optical assemblies 850 and 880,beam spots corresponding to the reflected light beams may be detectedand relative separation for each pair of reflected light beams can bedetermined. By determining if the different pairs of incident lightbeams are in focus with the object, such optical systems can determinean angle of the object with respect to the optical axis of the lens orany other reference axis. Thus, the information can be used to determinewhether the object is tilted or has changed orientation. Furthermore,multiple pairs of incident light beams may be used as a redundantmechanism for surface profiling or determining a working distancebetween the lens and the object. Although the optical assemblies 850 and880 show only two pairs of incident light beams, more than two pairs maybe used. In particular embodiments, it may be advantageous to use atleast 3 pairs of incident beams or to use at least 4 pairs of incidentbeams. The multiple focal regions may be aligned linearly or positionednon-linearly so as to define a two-dimensional geometric shape on theobject.

Information regarding the angle or orientation of an object can beprocessed by a system controller of an optical system to adjust theangle of the object. The angle can be adjusted to achieve a desired tiltor angle for an object. For example, the angle of a flat object can beadjusted so that it is closer to being orthogonal to the optical axis ofan objective lens used for imaging the object. Thus, an optical systemcan be configured to adjust an object to position a planar surface ofthe object to be orthogonal to the optical axis of a lens that is usedto image the surface.

FIGS. 17 and 18 are perspective and plan views, respectively, of anoptical assembly 502 formed in accordance with another embodiment. Theoptical assembly 502 may be used with various optical systems, such asthe optical system 100 shown in FIG. 1. As shown, the optical assembly502 includes an optical train 540 of optical components 541-545 thatdirect light beams along an optical track between an object of interest(not shown) and a focus detector 550. The series of optical components541-545 of the optical train 540 include a dual-beam generator 541, anobjective focus mirror 542, a conjugate lens 543, a beam combiner 544,and a beam folding device 545.

The optical assembly 502 includes a reference light source 512 thatprovides a light beam 528. In some embodiments, the light beam 528 mayfirst transmit through a collimating lens 546 (FIG. 18) and an opticalwedge 547 (FIG. 18). The optical wedge 547 may be rotated for fineadjustment of an optical path of the light beams. As shown in FIGS. 17and 18, the light beam 528 is incident upon the dual-beam generator 541,which provides a pair of parallel incident light beams as describedabove. The parallel incident light beams are directed toward the focusmirror 542.

The collimating lens 546 may be used to configure the incident andreflected light beams as desired. For example, the collimating lens 546may modify the light beam 528 so that the light rays of the incidentlight beams are not precisely parallel with respect to each other.Furthermore, the light rays may be modified so that the reflected lightbeams have a minimum diameter at the focus detector 550 when the opticalassembly 502 is in focus.

The dual-beam generator 541 directs the parallel incident light beamstoward the focus mirror 542. The focus mirror 542 reflects the incidentlight beams toward the conjugate lens 543. As shown, the focus mirror542 includes a pair of reflectors 551 and 553 (e.g., aluminized tabs)that are positioned to reflect the incident light beams and thereflected light beams from the object. The reflectors 551 and 553 mayfunction similar to a range limiter in that the reflectors 551 and 553may be sized to reflect only a limited range of reflected light beams.The focus mirror 542 is positioned to reflect the incident light beamsso that the incident light beams propagate parallel to an optical axis552 of the lens 543. The lens 543 may be a near-infinity conjugatedobjective lens.

As described above with respect to the optical assembly 202, thereflected light beams may propagate along a substantially equal oroverlapping optical path with respect to the incident light beamsthrough the lens 543 to focus mirror 542 which directs the reflectedlight beam toward the dual-beam generator 541. As shown in FIGS. 17 and18, the reflected light beams are incident upon and directed by thedual-beam generator 541 toward the beam combiner 544. In the illustratedembodiment, the beam combiner 544 is configured to modify a path spacingthat separates the reflected light beams. The path spacing at the beamcombiner 544 may be re-scaled to be substantially equal to theseparation distance of the reflected light beams at the focus detector550.

Like the optical train 240 (FIG. 2) described above, the optical train540 is configured to maintain a projection relationship between thereflected light beams throughout the optical track so that adegree-of-focus may be determined. Also shown in FIGS. 17 and 18, theoptical train 540 may include a beam folding device 545. The beamfolding device 545 functions to increase an optical path length betweenthe lens 543 and the focus detector 550. The beam folding device 545 mayincrease the gain and range of the optical assembly 502.

FIG. 19 is a side view of a beam folding device 545. The beam foldingdevice 545 includes a pair of spaced apart sides 560 and 562. The side560 includes an inlet window 561 that is sized to receive the reflectedlight beams from the beam combiner 544. The reflected light beams enterthe beam folding device 545 through the inlet window 561 and arerepeatedly reflected back and forth between the sides 560 and 562. Thesides 560 and 562 may be aluminized to reduce optical losses. Thereflected light beams may be transmitted through an outlet window 563and propagate to the focus detector 550 (shown in FIG. 17).

With each iteration in which the reflected light beams propagate betweenthe sides 560 and 562, an optical path length of the reflected lightbeams increases a width W of the beam folding device 545. The number ofiterations may be based upon an angle of incidence between the reflectedlight beams and the sides 560 and 562 and a length L of the beam foldingdevice 545. Accordingly, the beam folding device 545 may be sized,shaped, and oriented to provide an increase in the optical path length.Increasing the optical path length, in turn, may function as anamplifier to increase the gain and range of the optical assembly 502.

In the illustrated embodiment, the beam folding device 545 has anoptical body having a transparent material. However, in alternativeembodiments, the beam folding device 545 may include two opposingmirrors with ambient air therebetween. Furthermore, in the illustratedembodiment, the opposite sides 560 and 562 extend parallel to oneanother. In alternative embodiments, the beam folding device 545 mayinclude multiple sides or surfaces that may or may not extend parallelto one another. Optionally, the focus detector 550 may be affixed to thebeam folding device 545.

Returning to FIGS. 17 and 18, the optical assembly 502 may also includea pair of phase detectors 570 and 572. The phase detectors 570 and 572are positioned to receive respective portions of the reflected lightbeams that are transmitted through the beam combiner 544. The phasedetectors 570 and 572 may be used in optical systems that continuouslyscan an object when there is relative motion between the object andconjugate lens in a direction that is orthogonal (or perpendicular) tothe optical axis. The phase detectors 570 and 572 detect a phase of theintensity of the reflected light beams to determine, for example, anyphase differences that occur between the reflected light beams. Althoughthe exemplary systems in FIGS. 17 and 18 include a combination of aphase detector and a focus detector, it will be understood that eithertype of detector can be used absent the other.

FIGS. 20-22 illustrate an object 564 being scanned by the opticalassembly 502 (FIG. 17) while the object 564 is relatively moving in alateral direction (indicated by the arrow XD) with respect to the lens543 (FIG. 17). The lateral direction XD is orthogonal to the opticalaxis 552 of the lens 543. To move the object 564 relative to the lens543, the object 564 may be moved in the lateral direction XD by a stagecontroller and/or the lens 543 is moved in a direction opposite to thelateral direction XD. In particular embodiments, the optical assembly502 may use a differential phase detection mechanism for determining adegree-of-focus. The optical assembly 502 may alternatively oradditionally use a differential phase detection mechanism fordetermining the working distance between object 564 and lens 543. FIG.20 illustrates the object 564 being in focus with a focal region 556 asthe object 564 is moved in the lateral direction XD. If a surface 565 ofthe object 564 is located within a focal plane FP of the opticalassembly 502, the phase detectors 570 and 572 (FIG. 17) will detect asubstantially common phase for the reflected light beams 530B and 532B.

FIG. 21 illustrates a scan of the object 564 when the object 564 isbelow the focal region 556, and FIG. 22 illustrates a scan of the object564 when the object 564 is above the focal region 556. If the surface565 of the object 564 is below the focal plane FP as shown in FIG. 21,the phase detectors 570 and 572 will continuously detect a phasedifferential of the reflected light beams as the optical assembly 502scans the object 564. The phase differential indicates that the object564 is below the focal region 556 or focal plane FP. Similarly, if thesurface 565 of the object 564 is above the focal plane FP as shown inFIG. 22, the phase detectors 570 and 572 will continuously detect aphase differential of the reflected light beams 530B and 532B. The phasedifferential indicates that the object 564 is above the focal region556.

The object 564 in FIGS. 20-22 is scanned with the incident light beamsalong a substantially flat surface. However, in alternative embodiments,the object may include an array of microparticles or have a surface withrelief features such as regions of unevenness (e.g., ruled or groovedsurface). As the optical assembly 502 scans the surface of the object,the height or elevation of the detected surface may frequently changedue to the microbeads or uneven surface. Depending upon the movement ofthe object, one reflected light beam may be disturbed (e.g., incidentupon a microbead) before the other reflected light beam. In suchembodiments, relative positions of the microparticles or other surfacefeatures with respect to each other along the surface may also bedetermined.

FIG. 23 is a perspective view of a sample imager 600 formed inaccordance with one embodiment. The sample imager 600 may have similarfeatures, components, systems, and assemblies as described above withrespect to the optical system 100 and the optical assemblies 202 and502. As shown, the sample imager 600 includes an imager base 602 thatsupports a stage 604 having a sample holder 606 thereon. The sampleholder 606 is configured to support one or more samples 608 during animaging session. The samples 608 are illustrated as flow cells in FIG.23. However, other samples may be used.

The sample imager 600 also includes a housing 610 (illustrated inphantom) and a strut 612 that supports the housing 610. The housing 610can enclose at least a portion of an optical assembly 614 therein. Theoptical assembly 614 may include a focus assembly 616 and asample-detecting assembly 630. The focus assembly 616 may be similar tothe optical assembly 502 described above. For example, the focusassembly 616 may include an auto-focus line scan camera 620 thatreceives reflected light beams for determining a degree-of-focus of thesampler imager 600. The sample imager 600 may also include a filterwheel 622 and an alignment mirror 624 that directs light toward a sampledetector 632, which is shown as a K4 camera in FIG. 23.

FIG. 24 is a block diagram that illustrates a method 700 of determininga degree-of-focus of an object with respect to an optical assembly. Themethod 700 may be performed by various optical systems, such as thosedescribed herein. The method 700 includes providing at 702 a pair ofincident light beams to a conjugate lens. The conjugate lens may benear-infinite lens as described above with respect to the lenses 243 and543. The incident light beams can be directed to propagate through thelens parallel to an optical axis of the lens and are directed by thelens to converge toward a focal region. At 704, the incident light beamsare reflected by the object that is positioned proximate to the focalregion. The reflected light beams return to and propagate through thelens. In various embodiments, the method may include one or more mannersof determining a degree-of-focus of the optical assembly with respect tothe object based upon relative characteristics of the reflected lightbeams as indicated by three exemplary options 706, 708 and 710 shown inFIG. 24.

For instance, the method 700 may include determining at 706 a separationdistance that is measured between the reflected light beams. Forexample, the reflected light beams may be incident upon a detectorsurface and form beam spots thereon. The detector may communicate datarelating to the detected beam spots to a computing system, such as thecomputing system 120. The computing system may include a focus-controlmodule that analyzes the beam spots. For example, the focus-controlmodule may determine a centroid of each beam spot and then calculate aseparation distance measured between the beam spots.

In addition to (or alternatively) determining the separation distance ofthe beam spots, the method 700 may include analyzing at 708 a morphology(e.g., size, shape, and density) of each beam spot. The morphology ofeach beam spot may change due to the degree-of-focus of the opticalassembly or due to imperfections, such as dirt or bubbles, thatinterfere with the reflected light beams. For example, as describedabove, the beam spots may have different airy radiuses based uponwhether the object is in focus, below focus, or above focus.

In addition to (or alternatively) determining the separation distanceand morphologies of the beam spots, the method 700 may include at 710detecting a phase of each of the reflected light beams. A portion ofeach reflected light beam may be incident upon and detected by acorresponding phase detector. The phase detector detects a phase of thecorresponding reflected light beam.

The method also includes determining at 712 a degree-of-focus of theoptical assembly with respect to the object based upon at least one ofthe separation distance of the beam spots, the beam spot morphologies,and a comparison of the phase measurements. In particular embodiments,the degree-of-focus is only determined by the separation distance of thebeam spots. In other embodiments, the degree-of-focus is only determinedby comparing the detected phases of the reflected light beams.

At 714, the object may be moved in a direction toward or away from thelens based upon the degree-of-focus. For example, the object may bemoved to improve the degree-of-focus. In some embodiments, the object ismoved in a direction toward or away from the lens based upon therelative locations of the beam spots on a detector surface as describedabove. In some embodiments, the object is moved in a direction basedupon a comparison of the phase measurements.

In alternative embodiments, the operations of the method 700 may be usedto determine characteristics of a surface of an object. For example,various optical systems and assemblies may use the determined separationdistance or the detected phase measurements to determine a height ofsurface. For example, a height of various elements of a semiconductordevice that are mounted or deposited onto a surface of the device may bedetermined. Relative positions of the various elements may also bedetermined by scanning the surface at a predetermined rate.

FIG. 25 is a block diagram illustrating a control loop for controlling adegree-of-focus of an optical system with respect to an object. At 802,an imaging session may be started. At 804, the optical systemestablishes that an object, which may include an object as describedabove, is positioned near a focal region of a conjugate lens of theoptical system. The optical system may reflect incident light beams withthe object as described above.

Optionally, at 806, the optical system may determine reference values orstandards to facilitate determining whether the optical system is infocus. For instance, the object may be moved in a Z-direction toward andaway from the lens while detecting relative characteristics of thereflected light beams. The optical system can detect beam spots from thereflected light beams on a detector surface and record the relativepositions of the beam spots as the object is moved from an above-focusposition through the focal plane and into a below-focus position. Forexample, the beam spots may have a particular separation distance andspot morphologies when the object is located a known distance above thefocal plane, at the focal plane, and a known distance below the focalplane, respectively. The desired separation distance may also be basedupon (or a function of) dimensions of a dual-beam generator thatprovides parallel incident light beams or, alternatively, a beamcombiner that re-scales a path spacing of the reflected light beams. Theoptical system may also determine reference values or standards basedupon detected phase measurements of the reflected light beams asdescribed above.

At 808, the object is relatively moved to a new position and adegree-of-focus is measured at the new position. The optical system at810 may query whether the calculated degree-of-focus is sufficient. Ifthe calculated degree-of-focus is sufficient, the optical system movesthe object to a new position at 808. If the calculated degree-of-focusis not sufficient, the optical system queries at 812 whether the imagingsession has completed. For example, if the object has moved laterallybeyond the focal region, a null score may be determined by the opticalsystem. If the imaging session is not completed, the optical system at814 moves the object in a Z-direction toward or away from the lens. Thedirection may be based upon the relative characteristics determined bythe optical system. For example, if the relative locations of the beamspots indicate the object is below focus, the object may be movedvertically upward toward the focal plane. An amount of movement may alsobe a function of the relative characteristics. Optionally, the opticalsystem may re-determine the new degree-of-focus at the new Z-positionbefore moving to a new lateral position to confirm that thedegree-of-focus is sufficient. The optical system then moves at 808 theobject to a new lateral position.

FIG. 26 is a block diagram that illustrates a method 1000 of determininga working distance between an object and a conjugate lens of the opticalassembly. The working distance may then be used to determinedegree-of-focus or a surface profile of an object. The method 1000 maybe performed by various optical systems, such as those described herein.The method 1000 includes providing at 1002 a pair of incident lightbeams to a conjugate lens. The conjugate lens may be near-infinite lensas described above with respect to the lenses 243 and 543. The incidentlight beams can be directed to propagate through the lens parallel to anoptical axis of the lens. The lens directs the incident light beams toconverge toward a focal region. At 1004, the incident light beams arereflected by the object that is positioned proximate to the focalregion. The reflected light beams return to and propagate through thelens.

For instance, the method 1000 may include determining at 1006 a relativeseparation that is measured between the reflected light beams. Forexample, the reflected light beams may be incident upon one or moredetector surfaces and form beam spots thereon. The detector(s) maycommunicate data relating to the detected beam spots to a computingsystem, such as the computing system 120. The computing system mayinclude a module that analyzes the beam spots as described above. Inaddition to (or alternatively) determining the relative separation ofthe beam spots, the method 1000 may include analyzing at 1008 amorphology (e.g., size, shape, and density) of each beam spot.Furthermore, in addition to (or alternatively) determining the relativeseparation and morphologies of the beam spots, the method 1000 mayinclude at 1010 detecting a phase of each of the reflected light beams.A portion of each reflected light beam may be incident upon and detectedby a corresponding phase detector. The phase detector detects a phase ofthe corresponding reflected light beam.

The method also includes determining at 1012 a working distance of theoptical assembly with respect to the object based upon at least one ofthe relative separation between the reflected light beam, the beam spotmorphologies, and a comparison of the phase measurements. At 1014, acomputing system records the working distance at the particular locationwith respect to the object. At 1016, the computing system may determinea surface profile of the object based upon the working distancesdetermined by the optical system. Alternatively or additionally, acomputing system may determine an angle of the surface of the objectwith respect to the optical axis of the objective lens. The computingsystem can be further configured to instruct the optical system toadjust the relative angle between the surface of the object and theoptical axis to achieve a desired tip or tilt or orientation. Forexample, an adjustment can be made to position the surface of the objectto be orthogonal to the optical axis of the objective lens.

FIG. 27 is a block diagram illustrating control loops for operating anoptical system in accordance with various embodiments. At 1202, aprofile determining session of an object may be started. The object maybe, for example, a flow cell or a semi-conductor chip. At 1204, theoptical system may be calibrated to determine a reference relativeseparation that represents when the object is within the focal region.For example, the object may be imaged as the object is moved along thez-axis to and from the lens as described above. Image analysis maydetermine a reference degree-of-focus or reference working distance fromthe lens. In another embodiment, the lens may be replaced with a mirror.As such, the determined relative separation of the reflected light beamsmay function as a reference relative separation that identifies when theobject is within the focal region.

At 1206, the optical system may query a user as to which control loop toperform. A first control loop may be selected by the user. The firstcontrol loop may be an “open loop” system where the object holderpositions at 1208 the object at a predetermined z-position. At 1210, atleast one of the optical assembly and the object holder may be moved ina direction that is perpendicular to the optical axis of the lens. Inparticular embodiments, for example, those using multiple pairs ofincident beams such as shown in FIGS. 16A and 16B, the holder can bemoved to adjust the tip or tilt of the object. At 1212, the opticalsystem monitors the relative separation as the optical system scans thesurface. For example, the optical system may record a relativeseparation for a series of data points and associate each data pointwith a position along the surface. When the working distance decreases(i.e., when the height of the object surface increases) the relativeseparation may increase as shown in FIG. 9. When the working distanceincreases (i.e., when the height of the object surface decreases) therelative separation may decrease as shown in FIG. 7. Accordingly, asurface profile or topography of the object may be determined.

The user may also select a second control loop. The second control loopmay be a “closed loop” system where the optical system is configured tomove the object holder so that the object is maintained within the focalregion. At 1213, the object is positioned within the focal region. At1214, at least one of the optical assembly and the object holder may bemoved in a direction that is perpendicular to the optical axis of thelens. In particular embodiments, for example, those using multiple pairsof incident beams such as shown in FIGS. 16A and 16B, the holder can bemoved to adjust the tip or tilt of the object. At 1216, the opticalsystem monitors the relative separation as the optical system scans thesurface. When the object is moved out of the focal region or is nolonger sufficiently within focus, the optical system may move the objectin the z-direction to remain within the focal region. For example, ifthe relative separation changes from the reference relative separation,the object holder may move the object in a direction that is based uponthe changing relative separation. When the optical system moves theobject holder, the optical system records the distance that the objectwas moved. The recorded distance is indicative of the changing profile.When the working distance decreases (i.e., when the height of the objectsurface increases) the optical system moves the object away from thelens. When the working distance increases (i.e., when the height of theobject surface decreases) the optical system moves the object toward thelens.

Accordingly, during the first and second control loops, optical systemsdescribed herein may monitor the relative separation of the object asthe object is scanned. The optical system can record a change in workingdistance, which indicates a change in the surface profile or topographyof the object. At 1216, the optical system may determine a surfaceprofile based upon the changes in working distance.

FIG. 28 is a block diagram of a method 1100 of operating an opticalsystem, such as the optical systems described above. The method 1100includes providing at 1102 at least one pair of parallel incident lightbeams to a conjugate lens. The pair of parallel incident light beams maypropagate parallel to the optical axis of the lens or form anon-orthogonal angle relative to the optical axis. At 1104, the incidentlight beams are reflected by an object positioned proximate to a focalregion. The reflected light beams return to and propagate through thelens. The reflected light beams have a projection relationshipdetermined by the position of the object with respect to the focalregion.

The method 1100 also includes, at 1108, directing the reflected lightbeams with a plurality of optical components. The optical components maybe sized, shaped, and positioned with respect to each other to maintainthe projection relationship. For example, each optical component mayinclude one of a common planar surface that reflects both reflectedlight beams or first and second parallel surfaces that each reflects oneof the reflected light beams. At 1108, at least one of adegree-of-focus, a working distance, and a surface profile of an objectmay be determined based upon the projection relationship. Thedegree-of-focus, working distance, and the surface profile may bedetermined by a relative separation of the reflected light beams asdescribe above.

Accordingly, embodiments described herein may include methods andvarious optical systems and assemblies that control focus by reflectingincident light beams with an object and using relative positions,orientations, and characteristics of the reflected light beams todetermine a degree-of-focus or a working distance between the lens andthe object.

It is to be understood that the above description is intended to beillustrative, and not restrictive. For example, the above-describedembodiments (and/or aspects thereof) may be used in combination witheach other. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from its scope. While the specific components andprocesses described herein are intended to define the parameters of thevarious embodiments of the invention, they are by no means limiting andare exemplary embodiments. Many other embodiments will be apparent tothose of skill in the art upon reviewing the above description. Thescope of the invention should, therefore, be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. In the appended claims, the terms“including” and “in which” are used as the plain-English equivalents ofthe respective terms “comprising” and “wherein.” Moreover, in thefollowing claims, the terms “first,” “second,” and “third,” etc. areused merely as labels, and are not intended to impose numericalrequirements on their objects. Further, the limitations of the followingclaims are not written in means-plus-function format and are notintended to be interpreted based on 35 U.S.C. §112, sixth paragraph,unless and until such claim limitations expressly use the phrase “meansfor” followed by a statement of function void of further structure.

What is claimed is:
 1. A method for controlling a focus of an opticalsystem, the method comprising: providing a pair of incident light beamsto a conjugate lens, the incident light beams propagating parallel toeach other when received by the lens, the incident light beams beingdirected by the lens to converge toward a focal region; reflecting theincident light beams with an object positioned proximate to the focalregion, the reflected light beams returning to and propagating throughthe lens; determining relative separation between the reflected lightbeams; and determining a degree-of-focus of the optical system withrespect to the object based upon the relative separation, wherein theobject is in-focus when the reflected light beams exit the lens parallelto each other.
 2. The method according to claim 1 further comprisingmoving at least one of the lens and the object to adjust a relativeposition of the lens and the object with respect to each other when theobject is not in-focus, thereby improving the degree-of-focus.
 3. Themethod according to claim 1, wherein the providing operation includesproviding a single light beam to a dual-beam generator that provides thepair of incident light beams.
 4. The method according to claim 3,wherein the dual-beam generator has first and second parallel surfaces,the dual-beam generator reflecting and refracting portions of the singleincident light beam to generate the pair of incident light beams.
 5. Themethod according to claim 1, wherein the reflected light beams areseparated by a path spacing when exiting the lens, the method furthercomprising modifying the reflected light beams to re-scale the pathspacing to be substantially equal to the relative separation.
 6. Themethod according to claim 1, wherein determining the relative separationcomprises determining a separation distance between beam spots.
 7. Themethod according to claim 1, wherein the incident light beams arereflected by the object at multiple points along an optical axis of thelens, the method further comprising filtering the reflected light beamsso that the relative separation corresponds to a predetermined pair ofreflected light beams.
 8. The method according to claim 1 furthercomprising relatively moving the object in a direction that issubstantially perpendicular to the optical axis of the lens, therelatively moving operation including moving at least one of the objectand the conjugate lens, wherein the determining operation includescontinuously monitoring the relative separation.
 9. The method accordingto claim 1, wherein the reflected light beams exit the lens andpropagate along an optical track between the lens and a detector, theoptical track having a length that is configured to facilitatedetermining the relative separation.
 10. The method according to claim 9wherein a beam folding device redirects the reflected light beams frommultiple surfaces to increase the length of the optical paths of thereflected light beams.
 11. The method according to claim 1, wherein thelens has an optical axis extending therethrough, wherein the incidentand reflected light beams that propagate through the lens aresubstantially equally spaced apart from the optical axis.
 12. The methodaccording to claim 1, wherein the object is a sample comprisingbiological or chemical substances, the optical system performing one ofepi-fluorescent imaging and total-internal-reflection-fluorescence(TIRF) imaging of the sample.
 13. The method according to claim 1,wherein the object is a sample comprising biological or chemicalsubstances, the optical system performing time-delay integration (TDI)analysis of the sample.
 14. The method according to claim 1, wherein theobject is a sample comprising biological or chemical substances, thesample including a flow cell.
 15. The method according to claim 1,wherein the pair of incident light beams is a first pair of incidentlight beams, the providing operation further comprising providing asecond pair of incident light beams to the conjugate lens, the incidentlight beams of the second pair propagating parallel to each other whenreceived by the lens, the first pair of incident light beams propagatingthrough the conjugate lens at a first angle with respect to an opticalaxis and the second pair of incident light beams propagating through theconjugate lens at a different second angle with respect to the opticalaxis.
 16. An optical system comprising: a reference light sourceconfigured to provide a pair of incident light beams; a conjugate lenspositioned to receive the incident light beams propagating parallel toeach other, the lens directing the incident light beams toward a focalregion; an object holder configured to hold an object with respect tothe focal region, the object reflecting the incident light beams so thatthe reflected light beams return to and propagate through the lens; anda focus-control module configured to determine a relative separationbetween the reflected light beams and determine a degree-of-focus basedupon the relative separation, the object being in-focus when thereflected light beams exit the lens parallel to each other.
 17. A methodfor determining a working distance of an object from a conjugate lens,the method comprising: providing a pair of incident light beams to theconjugate lens, the incident light beams propagating parallel to eachother when received by the lens, the incident light beams being directedby the lens to converge toward a focal region; reflecting the incidentlight beams with the object positioned at an working distance from thelens proximate to the focal region, the reflected light beams returningto and propagating through the lens, wherein the object is above thefocal region when the reflected light beams exit the lens diverging awayfrom each other; determining relative separation between the reflectedlight beams; and determining the working distance between the object andthe lens based upon the relative separation.
 18. An optical systemcomprising: a reference light source that is configured to provide apair of incident light beams; a conjugate lens positioned to receive theincident light beams, the incident light beams propagating parallel toeach other when received by the lens, the lens directing the incidentlight beams to a focal region; an object holder that is configured tohold an object at a working distance from the lens, the objectreflecting the incident light beams so that the reflected light beamsreturn to and propagate through the lens, wherein the object is abovethe focal region when the reflected light beams exit the lens divergingaway from each other; and a computing system that is configured todetermine relative separation between the reflected light beams anddetermine the working distance based upon the relative separation.
 19. Amethod of operating an optical system, the method comprising: providingparallel incident light beams to a conjugate lens, the incident lightbeams propagating parallel to each other when received by the lens, theincident light beams being directed by the lens to converge toward afocal region; reflecting the incident light beams with an objectpositioned proximate to the focal region, the reflected light beamsreturning to and propagating through the lens, the reflected light beamshaving a projection relationship; and directing the reflected lightbeams with a plurality of optical components along an optical track, theoptical components being shaped and oriented to maintain the projectionrelationship of the reflected light beams; determining a degree-of-focusbased upon the projection relationship, wherein the object is in-focuswhen the reflected light beams exit the lens parallel to each other.